IV. Combinatorial Chemistry
1. Library synthesis
a) in solution, parallel synthesis b) on solid support c) split and combine, one bead one compound
2. Deconvolution and Tagging
3. Dynamic Combinatoric Chemistry
Parallel Library Synthesis
• 12 reactions provide 9 compounds • The library members are spatially separated, so this technique can be used for solution as well as solid phase synthesis
diversification divide diversification reaction reaction Split-pool Synthesis
• 6 reactions lead to 9 compounds • Each library member must be compartmentalized (each compound on its own bead) to allow pooling of the library
splitdiversification pool and split diversification reaction reaction
An Example of Split-Pool Synthesis
O NHBoc NH 1) Pd2dba3, Cl Me 2 O O SnMe O 3X 3 2) TFA 3X Me
NH2 O O O 3X Me NH2 Cl NHBoc NH2 NHBoc 1) Pd2dba3, O O O 3X O O SnMe O SnMe 3X 9X 3 3X 3 2) TFA
NH2 O OCl O 3X Cl NH2 Cl NHBoc 1) Pd dba , 2 3 O O 3X 3X O SnMe3 2) TFA Cl
splitdiversification pool reaction
Ellman, J. et al. J. Am. Chem. Soc., 1995, 117, 3306. O NH2 NH2 NH O O O O O NH 1) O NH 2 F O 2 NH NH Me NH 2 NHFmoc O O NH2 O O O O O Me O 2) piperidine Cl Cl
HO2C
NH2 O CO H NH2 O O 2 HO C O NH HO2C 2 1) F O O O O NH2 NH2 NHFmoc NH2 Me NH NH O NH2 O O O 2) piperidine O O Me O O Cl Cl
NH2 S O S O O O 1) NH F 2 S NH2 S NHFmoc NH O NH2 O O O O NH O NH 2 Me O 2 NH O 2) piperidine Me NH Cl O O O O split diversification Cl reaction
Overview of the Entire Split-Pool Library
R2 O 2 O R NHFmoc NH F NHBoc Pd dba , 2 2 3 TFA NHFmoc NH O O O O SnMe O 3 1 35 Amino Acids O R 1 Cl R1 R 20 Acid 20 cpds 20 x 35 = 700 cpds Chlorides
R3 H O O N N 1) piperidine Base, R3X TFA R2 R2 2) AcOH N N O 16 Alkylating O R1 Agents R1 700 cpds 20 x 35 x 16 = 11,200 cpds
Split-Pool step:
Ellman, J. et al. J. Am. Chem. Soc., 1995, 117, 3306. Example of the Efficiency of the Split-pool Strategy
• Optimization of 154 reactions affords 105 amplification in the number of compounds R1 R1 I
O H R2 N 1 O H 2 NH R R NH2 O O N H O N N O O H 62 amines H 30 alkynes N HO O H O H N O O O O 18 iodides O R1
R2 O NH
R3 OH O N 3 O R O H N 30 + 62 + 62 = 154 reactions 62 acids 18 X 30 X 62 X62 = 2.1 million compounds O O O
Schreiber SL et al. JACS, 1998, 120, 8565.
Structural Characterization: Direct Methods
Off-bead Analysis • Cleavage, then use of analytical techniques used in TOS (e.g. LC, MS, NMR) • Requires high sensitivity and high throughput format
•Example: LC-UV/ MS OH OH S O Ph HO N Ph Structural Characterization: Direct Methods
On-bead Analysis I • Can be used to monitor the progress of a reaction • MAS-NMR ( Magic angle spinning NMR ) is necessary due to polymer
Magic angle rotor (left), rotor spinning at the magic angle (right)
MAS- NMR spectrum (600 MHz)
Si O OMe O
O O O O O
O
Structural Characterization: Direct Methods
On-bead Analysis II • Example: Single-bead FT-IR microspectrometry • Can be used to monitor the progress of a reaction
OO OO H O HO O O O DIC, DMAP, DMF
Beads in IR cell
Wavelength (cm-1) Structural Characterization: Indirect Methods
Deconvolution • Screen as a mixture of compounds then re- synthesize and re-assay possible candidates in active pools
Drawbacks: • Interference by unwanted properties of other compounds (e.g. cytotoxicity) • Possible synergistic interaction of multiple compounds • Sub-library synthesis is cumbersome
Encoding
• Encoding should provide a fast and simple way to identify the structure of all library members • Classification – Spatial encoding: position of the compound provides the information about its structure (possible only in parallel synthesis) – Graphical encoding: bar codes or other graphical tags are displayed on the solid support used in the library synthesis – Chemical encoding: every reaction used in the library synthesis is recorded on the solid support by the chemical attachment of a tag • binary coding (presence or absence of a tag) or polymer based (polypeptide, DNA) – Spectrometric encoding: using a spectrometric technique (NMR, MS, Fluorescence microscopy, NMR etc.) to read tags directly from the solid support – Electronic encoding: radio frequency memory chip attached to the solid support records and emits coded information
A. C. Czarnik Current. Opp. Chem. Biol. 1997, 1, 60 Encoding in Split-pool Synthesis
• Optimization of 6 reactions leads to 9 compounds • Each library member must be isolated on its own bead to allow pooling of the library How do you know what compound is on a given bead after a pool step?
splitdiversification pool and split diversification reaction reaction
Chemical Encoding in Split-Pool Synthesis
• Every diversification reaction is followed by a tagging reaction in which a tag(s) that codes for a particular transformation is covalently attached to the solid support Decoding
• Every bead has tags that provide information, once cleaved, about the chemical history of that bead • Conditions for cleavage of compound and tags have to be orthogonal
compound cleavage tag cleavage
small molecule
Binary Chemical Coding
11 10 011101 110 111 1 Building blocks
Binary 00 01 001 010 100 0 (base 2) codon
Tags
2 digit codon 3 digit 1 digit 22 = 4 max
11 110 1 n binary tags code for 2n building blocks Another Example
10 011 0
11 10 011101 110 111 1 Building Blocks
00 01 001 010 100 0
Tags
Halogenated Aromatics As Tags
• Small amount of tag can be reliable detected (0.5-1 pmol/bead) using easily automated electron capture GC in the mixture of tags based on different retention times • Inert under most reaction conditions
OMe Cl OO X n X=Cl or H N HC 2 Cl Cl O X
linker variable variable region electrophore
W. C. Still et al. J. Org. Chem. 1994, 59, 4723 Attachment and Cleavage of Tags
• Tags are attached using rhodium carbene-insertion chemistry and
can be cleaved using (NH4)Ce(NO3)6 (CAN)
Cl HF O (CH2)n O X cleavable
N2HC OMe Cl Cl Me MeMe X O TAG Me Si X = H or Cl * O small molecule MeMe Me Me n = 1-14 Si O * O small molecule X Rh2(O2CC(Ph)3)4 OMe Cl Cl
CH2Cl2, 25 °C O (CH2)n O X Cycloheptatriene Cl TAG CAN cleavable
Binary Chemical Encoding of a Peptide Library
• A library of decapeptides was synthesized and screened for binding to 9E10 mAb. • 7 Amino acids were used at each position (S, I, K, L, Q, E, D) • Every amino acid was assigned a 3 digit binary codon (001=S, 010=I, 011=K, 100=L, 101=Q, 110=E, 111=D) where 1=presence of a tag and 0=absence • For each step in the library synthesis there are 3 tags designated nX (total of 18 tags for a library of 117,649 members, maximum encodable is 218 = 262,144) EQKLISEEDL known to bind 9E10 H2N-X-X-X-X-X-X-E-E-D-L-G-G-G-G-
OO O HO2C n Arx O NO2
linker electrophoric tag
nX general formula of tags used
110 101 011 100 010 001 identified as the best binder
W.C. Still et al. PNAS 1993, 90, 10923 Dynamic Combinatoric Chemistry
Virtual Dynamic Combinatoric Library Virtual Dynamic Combinatoric Library - macrocycles
Virtual Dynamic Combinatoric Library – carboanhydrase inhibitors
Lehn et al. 2106 Virtual Dynamic Combinatoric Library
Lehn et al. 2106
Virtual Dynamic Combinatoric Library – metal grids
Lehn et al, PNAS, 2003, 100, 11970 Virtual Dynamic Combinatoric Library – metal grids
Lehn et al, PNAS, 2003, 100, 11970
Virtual Dynamic Combinatoric Library – neuraminidase inhibitors
Eliseev et al, PNAS, 2002, 99, 3382 Virtual Dynamic Combinatoric Library – neuraminidase inhibitors
Eliseev et al, PNAS, 2002, 99, 3382
Virtual Dynamic Combinatoric Library – acetylcholine esterase inhibitors
Sharpless et al, PNAS, 2004, 101, 1449 Bourne, Yves et al. (2004) Proc. Natl. Acad. Sci. USA 101, 1449-1454
Pseudodynamic Dipeptide Library
Protease from Streptomyces griseus
Angew. Chem. Int. Ed. 2004, 43, 2432. Pseudodynamic Dipeptide Library
Angew. Chem. Int. Ed. 2004, 43, 2432.
Pseudodynamic Dipeptide Library
CA = carbonic anhydrase
Angew. Chem. Int. Ed. 2004, 43, 2432. V. Diversity Oriented Synthesis
Convergent Synthesis – Target oriented synthesis (TOS)
• complexity-generating Synthesis planning: rxns. retrosynthetic • fragment coupling rxns. one analysis molecule
Divergent Synthesis – Diversity oriented synthesis (DOS)
• complexity-generating rxns. many Synthesis planning: • multicomponent coupling different forward synthetic • diversity-generating rxns. molecules analysis
Molecular Complexity
TOS: Retrosynthetic Analysis of Saframycin A
OMe OMe OMe HO Me O Me HO Me OMe H O H OMe H H H H OMe Me OMe Me O Me N Me N Me N Me NH N N MeO MeO H MeO H H N OH NC O CN OH CN NH FmocHN NHFmoc O O O Intramolecular 2 pictet-spengler reactions Strecker Reaction Me saframycin A OMe OMe H HO Me Me CHO OH OMe OMe H NH NH Me OMe MeO 2 N OH OHC Me H N H MeO 2 H OMe HCN OH NC N HN CHO O O FmocHN Imine formation and Strecker reaction A. G. Myers and D. W. Kung J. Am. Chem. Soc. 1999, 121, 10828. Pseudoephedrine as a Chiral Auxiliary for Enolate Alkylation
OMe H Me CHO
NHFmoc MeO OTBS
1. LHMDS, LiCl, 0ÞC Me O Me O THF LiH2N-BH3 NH 2 NH2 N 2. OMe N THF OH Me Me Br OH Me OTBS 0ÞC 80% yield pseudoephedrine MeO MeO OMe OTBS Me 89% de H
NH2 NHFmoc NHFmoc HO HO Dess-Martin O Fmoc-Suc Periodinane OTBS OTBS OTBS NaHCO3 CH2Cl2 MeO OMe MeO OMe MeO OMe Me Me Me
A. G. Myers, P. Schneider, S. Kwon, D. Kung J. Org. Chem. 1999, 64, 3322. TOS: A Concise Synthesis of Saframycin A
OMe 96% ee OMe TBSO Me H Me CHO OH OMe H OMe Me OMe NHR NH Na2SO4 LiBr MeO H 2 N NC OH Me CH2Cl2 RHN DME H 23ÞC MeO H H 35ÞC N OMe 90% OTBS NC N protected 65% 92% ee Fragment aldehyde O O coupling
OMe OMe HO Me HO Me OMe H OMe H H H Me OMe Me OMe N X TBAF, HOAc N Me then DBU, 23ÞC RHN H2N MeO H H 92% MeO H H N OTBS NC OH NC N
O O X = H O CH O, NaBH(OAc) 2 3 X = Me 94% O
R = Fmoc = Myers and Kung JACS, 1999, 122, 10828.
TOS: A Concise Synthesis of Saframycin A
OMe OMe HO Me CHO HO Me OMe H FmocHN OMe H H H Me OMe Na2SO4 Me OMe N Me N Me CH Cl H2N 2 2 NH MeO H H 23ÞC MeO H N OH NC 66% OH NC N FmocHN O O
OMe OMe HO Me O Me OMe H 1. DBU O H H H Me OMe 2. ClCOCOCH3, Me O ZnCl2, TMSCN N Me PhNEt2 N Me TFA, THF, 23ÞC N 3. PhIO, MeCN- N MeO H MeO H 86% H20 OH CN 52% O CN NHFmoc NH O O Me saframycin A Myers and Kung JACS, 1999, 122, 10828. Why Perform Diversity-Oriented Synthesis?
• To systematize the discovery of molecules with properties that are difficult to design or predict.
• DOS is especially useful in cases where processes are complex and few of the “design”criteria are well understood. For example: • Asymmetric catalysis. • Small molecules with specific biological functions (protein binding, cellular effects).
• DOS approaches are useful when molecules that act through new mechanisms are desired. This is useful because it may illuminate aspects of chemistry or biology that have not yet been illuminated. It is possible because the screen may make no assumptions about mechanism. For example, a DOS approach could be used to discover Diels-Alder catalysts that work by an unexpected and completely novel mechanism and in the process illuminate new means of promoting cycloaddition reactions.
DOS: Synthesis, Screen, Discover, Study
CO2H NH 2 N O OH
S NH
Me Me HN NH NN 2 Me Me CO2H Screen for molecule(s) NN N with desired properties CO2H MeO SH O N OEt MeO SH Me O HO O CHO
Me O O Discovery of molecule with desired properties NH2 Synthesis of diverse molecules: Molecules may be biased towards properties that will achieve the goal (e.g. protein-binding elements or metal binding elements
Study Mechanism; understand chemistry or biology DOS vs. TOS in Enantioselective Catalysis (Example 1)
OH acylation OAc OH catalyst Me RR Ac2 O
racemic
Me N 2 Target-Oriented Synthesis Approach Me Me N Fe Ph Ph H H P Ph Ph Ar Ph Me 9 steps; 7 steps; separation by chiral HPLC; not easily modified; not easily modified limited scope of benzyl alcohols; S = 43-65 with benzyl alcohols; S = 15-389! S = 14-22 with allylic alcohols E. Vedejs, O. Daugulis G. Fu Acc. Chem Res . 2000 , 33, 412. J. Am. Chem. Soc. 1999 , 121 , 5813. J. Ruble, H. Latham, G. Fu J. Am. Chem. Soc. 1997 , 119, 1492.
DOS vs. TOS in Enantioselective Catalysis (Example 1)
OH acylation OAc OH catalyst Me RR Ac2 O
racemic
Diversity-Oriented Synthesis Approach
NMe N O iPr O O iPr O H H H H N N N N BocHN N N N OMe H H H O O O iPr O Me CONHR NR
16 steps-easily synthesized via amide coupling reactions on solid phase; structure easily modified;
G. Copeland and S. Miller J. Am. Chem. Soc . 2001 , 123, 6496. An Example of DOS: Discovery of New Enantioselective Acylation Catalysts for Kinetic Resolution
biasing element OAc NMe N for acylation Me 500μM OH catalytic H Me bead O library N H BocHN AA6 AA5 AA4 AA3 AA2 AA1 N Me OH N N Ac2O O H O H PhCH3 Me N 25ÞC D-Val, D-Phe, D-Pro, L-Ile, L-(OtBu)-Tyr, L-(trt)-Gln D-Ala, L-(trt)-Asn, Gly, Aib, L-(OtBu)-Asp, L-(Boc)-Trp L-(trt)-His, D-(OtBu)-Glu fluorescence detector of AcOH 100,000 of 146 (7.5 million) catalysts made
NMe select N O iPr O O iPr O H H H H brightest N N N N BocHN N N N OMe H H H beads O O O iPr O Me CONHR NR
Fluorescent beads= Krel=KR/KS=20 active catalysts (95% ee at 48% conv)
Copeland and Miller J. Am. Chem. Soc. 2001, 123, 6496.
Plan for Discovery of Enantioselective Peptide-Based Acylation Catalysts
π-Stack π-Stack
O O other other R Gen. Base R Gen. Base N functionality N functionality H H H O H-Bond versus H O H-Bond NUC O R2 NUC O R2 O R1 O R1 R R Me O Me O TS1 TS2
Include Amino Acids with Desireable Properties, but use Approach that is Modular and Variable
OH OAc
R1 R2 R1 R2
Ac2O
Non-Fluorescent Fluorescent catalyst catalyst H N N H H N N
O O Brightest Beads Contain Most Active Catalysts Split-Pool Diversity-Oriented Synthesis of Acylation-Biased Peptides
biasing element NMe N for acylation 500μM H Me bead N H O BocHN AA6 AA5 AA4 AA3 AA2 AA1 N N N O H H O N D-Val, D-Phe, D-Pro, L-Ile, L-(OtBu)-Tyr, L-(trt)-Gln D-Ala, L-(trt)-Asn, Gly, Aib, L-(OtBu)-Asp, L-(Boc)-Trp L-(trt)-His, D-(OtBu)-Glu fluorescence detector of AcOH 1 AA in each of 16 different flasks R 500μM 500μM Me bead OH H O FmocHN O Me bead O N H H N O FmocHN N 2 N N N O H i H H N HBTU, Pr2NEt R O N DMF, 25ÞC
500μM N O Me bead H O split into 16 flasks pool into one flask H H2N N repeat 6 times N N DMF H H R O N
NMe N
OH NMe 500μM BocHN N Me bead 500μM H H2N O H Me bead AA6 AA5 AA4 AA3 AA2 AA1 N N H N HBTU, iPr NEt BocHN AA AA AA AA AA AA N H 2 6 5 4 3 2 1 N O DMF, 25ÞC O H O
Discovery of Powerful Enantioselective Acylation Catalysts
cat. OAc OH Ac2O, PhMe, -65ÞC Me Me krel = 20
OAc OH cat. Me Me Ac2O, PhMe, -65ÞC
krel = 50
OAc OH cat. Ac O, PhMe, -65ÞC 2 Me Me krel = 9.0
cat. OAc OH Ac O, PhMe, -65ÞC 2 Me Me Me Me krel = 4.0
NMe N O iPr O O iPr O H H H H cat. = N N N N BocHN N N N OMe H H H O O O iPr O Me CONHR NR
This peptide-based catalyst would have been impossible to design - DOS leads to a discovery. Use of a DOS-Catalysis Approach in Complex Molecule Synthesis
O IMes N N IMes NH2
O O O O Cl2Ru O O O OH O Ph O O H2N OMe Cy3P LiAlH4
Me N NH CH2Cl2, 55ÞC Et2O N N N O O O O t mitomycin C O Bu OtBu OtBu O OH O OAc O OH O O O O R R 1 2 2 mol% CAT N R3 + HN Me H O Ac2O N PhCH N N N HN 3 O O O O NH OtBu OtBu OtBu N (Xaa)n BOC S = 9.8 = OOMe 90% ee @ 53% conversion DOS and Screen of 152 peptides NMe N O iPr O O iPr O O Ph H H H H N N N N BocHN N N N OMe N H H H Me N H O O O iPr O Me O CONHR N O HN NR NH O Ph N BOC previously optimized catalyst only produces S = 5.0 NH Ph CO Me CAT 2 S. J. Miller et al. Organic Letters; 2001; 3(18); 2879-2882
Enantiospecific Synthesis of a Mitomycin Core Structure
O NH2 O O H2N OMe
Me N NH O mitomycin C
O OH O O O O 1. Oxone, NaHCO 3 O acetone-H O OMe 2 HNO3,MeOH Sm(OiPr) 3, TMSN 3 O 2. (ClCO) 2, DMSO 81% N O N N CH2Cl2 Et3N, CH2Cl2 O 86% 85% O OtBu OtBu
O O O OMe OMe resin bound PPh OMe OH MsCl, Et3N OMs 3 N N CH2Cl2 iPr2NEt, THF-H 2O N NH N3 N3 42%, 2 steps
. S. J. Miller et al. Org. Lett 2001 DOS vs. TOS in Enantioselective Catalysis (Example 2)
O O Target-Oriented Synthesis Approach cat. Cu Salt cat. ligand
R2Zn Me n n O R PN n = 1-3 O Me
98% ee with wide range of 6-membered rings however, low ee for 5 and 7-membered rings; the structure is not easily modified B. Feringa et al. Angew. Chem. Int. Ed. Engl. 1997, 36, 2620.
Difficult Substrates O O
Me
Me 10% ee 53% ee
Ligand structure optimization Is difficult and time consuming
DOS vs. TOS in Enantioselective Catalysis (Example 2)
O O cat. Cu Salt Diversity-Oriented Synthesis Approach cat. ligand Me Me O R2Zn H n n N R N NHBu n = 1-3 O PPh2 Ph biasing element for metals
72-98% ee with wide range of 5,6, and 7-membered rings 3 steps-easily synthesized via amide coupling reactions; structure easily modified; S. Degrado, H. Mizutani, and A. Hoveyda J. Am. Chem. Soc. 2001, 123, 755.
Difficult Substrates O O O
Me Me Me Me Me 72% ee Me 62% ee 79% ee
Rapid ligand optimization using DOS approach The DOS approach allows optimization
O O cat. Cu Salt cat. ligand
R2Zn n n n = 1-3 R positional optimization strategy
Me Me O AA1 H Me AA2 Me N Me Me Me Me N NHBu O AA3 O H H O N OMe N PPh2 Ph N N N NHBu H O O O PPh2 PPh2 Difficult Substrates OtBu OtBu O O O O O O Me Me Me Me Me Me Me 72% ee Me 62% ee Me Me 79% ee 91% ee Me 81% ee Me 85% ee
rapid ligand structure optimization The positional optimization strategy involves optimizing because it was discovered using DOS approach each AA individually while holding the others constant
S. Degrado, H. Mizutani, and A. HoveydaJ. Am. Chem. Soc. 2001, 123, 755.
Use of DOS to Discover Enantioselective Catalysts of the Strecker Reaction (Example 3)
O
N 1. catalyst F3C N 2. TFAA H + HCN * CN
RR' Ph Ph O O P N N M P N N M Ph M O O Ph R R
binap bisoxazoline salen These ligands that are common in asymmetric catalysis are not well suited for a DOS approach since: 1. They do not provide a nonobtrusive site for attachment to the solid-phase 2. They are not condusive to wide structural variations (diversity) Use of DOS to Discover Enantioselective Catalysts of the Strecker Reaction (Example 3)
RR' Ph Ph O O P N N M P N N M Ph M O O Ph R R
binap bisoxazoline salen These ligands that are common in asymmetric catalysis are not well suited for a DOS approach since: 1. They do not provide a nonobtrusive site for attachment to the solid-phase 2. They are not condusive to wide structural variations (diversity)
Metal-binding library plan RR' RR' easily varied structure; high yielding synthesis O N linker amino linker N N 1 acid 2 M M O diversity by varying O R'' R, R', R'', linkers and amino acid R'' tridentate Schiff biasing element base complex to bind metals Sigman and Jacobsen JACS, 1998, 120, 4901.
Parallel Synthesis of Schiff-Base Ligands on the Solid-Phase
1 R NO O 2 HO 1 NO2 NHFmoc O R1 O R O O 1. H Cl O H O N N NH2 N NH N N O N HBTU, HOBT, 2 i H 5 H H 5 Pr2NEt, CH2Cl2-THF O H 5 i O Pr2NEt, DMF 2. 30% piperidine CHO R2 HO R2 H N 2 2 2 O R1 O R O R1 O R H 3 4 H NH2 N R2 R R N R2 N N N N N N Et3N, DMF H 5 H H DMF H 5 H H O NH2 O N HO
N N R3 R4 HBTU = N PF6 HOBT = N N N NMe O 2 OH
NMe2 Initial Screening Identifies a Metal-Free Catalyst
O
N 1. catalyst F3C N 2. TFAA H + HCN * CN
Library 1
O O R H 2 N R N N N 2 metal None Ti Mn Fe Ru Co Cu Zn Gd Nd Yb Eu H 5 H H O N ee 19 4 5 10 13 0 9 1 2 3 0 5 conversion 59 30 61 69 63 68 55 91 95 84 94 34 HO
tBu tBu
library size = 12 compounds Although the library was based upon a Schiff base that would bind metals, the most enantioselective catalyst is the metal-free ligand!
Library #2: Screen metal-free ligand but vary amino acid, diamine, and salicyaldehyde
Synthesis and Screening of Metal-Free Catalysts
Library 2 R3-salicylaldehydes O R1 O R H 2 2 R -diamines R3 R4 N R2 N N N R1-amino acids t t H 5 H H A Bu Bu O N B tBu H H2N H2N Ph t HO Leu, D-Leu C H Bu His, Phg (phenyl glycine) D tBu OMe H2N H2N Ph 3 4 E Br Br R R t F Bu NO2 library size = 48 compounds (6 salicylaldehydes x 4 amino acids x 2 diamines): made in 48 flasks simultaneously- parallel diversity-oriented synthesis
test all 48 compounds in individual Strecker reactions 5% ee if D-Leu
if S = NH2, 21% ee
O O H O S N H H N N N N N H 5 H H N N N N O N H H H H O N O N HO 32% ee 30-45%ee HO 45-55%ee HO tBu R4 tBu tBu tBu tBu 4 t R = Bu, H, or OMe better ee without linker thiourea improves ee Winning Catalyst Would Not Have Been Found By One-At-A-Time Approach
Library 3 R1 S R2 R1-amino acids 2 H R -diamines X-salicylaldehydes N R2 N N H H Leu, Ile, Met, Phe OMe O N t t H N Ph Tyr (O Bu), Val, Thr, (O Bu), H2N 2 H HO Nor (norleucine), Phg tBu Chg (cyclohexylglycine, H2N H2N Ph Br tBu X t-Leu (tert-Leucine) H2N N Ph H2N library size = 132 compounds (4 salicylaldehydes x 11 amino acids x 3 diamines): made in 132 flasks simultaneously- parallel diversity-oriented synthesis
S H Ph N N N H H O N
HO
tBu OMe
Winning Catalyst
A Parallel Combinatorial Approach to Catalyst Optimization
90% ee 80%
70%
60%
50%
40%
30%
20%
CH H 10% CH OMe CH Br CH t-Bu DP H 0% DP OMe DP Br Ile Val DP t-Bu Chg Nor t-Leu Leu CP H Tyr CP OMe Met Thr Phe CP Br Phg MS Sigman, EN Jacobsen CP t-Bu JACS 1998, 120, 4901 Diamine/Aldehyde Amino Acid NMR Solution Structure of the Optimized Strecker Catalyst
t-Bu
HO OCOt-Bu t-Bu N H HN NPh N O O H
EN Jacobsen and N Zondlo
NMR Solution Structure of the Imine-Catalyst Complex Enantioselective Synthesis of α-Amino Acids
S H Ph N N N H H O N
N N 2 mol% HO O O or O R H R H F3C N F3C N HCN, PhCH3, -70ÞC tBu O tBu or R = wide range of aromatic R CN R CN and aliphatic groups then TFAA 77-95% ee 70-98% yield O O 1. HCl (conc.) NH HCl H NPh65% H2SO4, 45ÞC H NPh2. H2, Pd/C, MeOH 2
CN CO2H CO2H
99% ee 84% y.
M. Sigman, P. Vachl, and E. N. Jacobsen Angew. Chem. Int. Ed. Engl. 2000, 39, 1279.
Catalytic Enantioselective Synthesis of Quaternary Centers
O H Ph N N N H H O N 2 mol% HO O H HCN, PhCH , -70oC tBu O tBu N 3 Me N R CN R Me R = aromatic and some aliphatic groups
P. Vachl, and E. N. Jacobsen Org Lett. 2000, 2, 867. Split-Mix Diversity-Oriented Synthesis of a 1,3-Dioxane Library
O OH O 500 µm PS N O bead H O OH OH Si SR2 i-Pr i-Pr
O R1 O OH O 2 2 OH OH 500 µm R SH or R NH 2 PS N O NR bead H i i O Me PrOH, Pr2NEt Si O R1 i-Pr i-Pr 50ÞC O OH O 90 diols; 500 µm PS N O 30 reactions bead H O Si i-Pr i-Pr
pool then split into 30 vials (each vial contains equal amounts of the three epoxyols)
react with 30 S. Sternson, J. Louca, J. Wong, S. Schreiber different 2Þ amines JACS 2001, 123, 1740. or thiols
Building Blocks Used in the 1,3-Dioxane Library
OH OH OH O (a) O O
HO HO HO Me (±)
(±) (b) H H Me N N HN HS HN OH N N HS N Me HO HN N NSH N N OH SH
SH Me SH H H Me Cl O N S N NH H 2N SH N + SH O N N N N N HN O - Cl OH
SH NH N SH SH N O N Me OH OH Me N N HN HO NH OH NN HN Me NSH HN Me COOH MeO OMe OH O
HN N Cl H N O O O O NH SH N OH Me N O O HN N N HN O H H O Split-Pool Diversity-Oriented Synthesis of a 1,3-Dioxane Library
NH NH2 2
NHFmoc
NHFmoc OO OO 2 OH OH or SR2 NR SR2 1 O R1 O R O R1 MeO OMe MeO OMe NH2 NH2 OH OH HCl, TMSCl NR2 O then O R1 OO OO N 2 SR2 NR H 1 90 diols; O R1 O R 30 reactions 180 diols; pool, then split 32 reactions into 2 vials (90 diols in each of 2 vials) 1 or R = H or Me react with 2 different acetals
S. Sternson, J. Louca, J. Wong, S. Schreiber JACS 2001, 123, 1740.
Split-Pool Diversity-Oriented Synthesis of a 1,3-Dioxane Library
H H 3 NH2 3 N R NH2 N R O O
OO OO OO OO 2 2 SR2 NR SR2 NR 1 1 O R1 O R O O R1 O R H 3 H 3 3 NH2 NH2 Cl R N R N R
DMAP O O
OO OO OO OO 2 2 SR2 NR SR2 NR 1 1 O R1 O R O R1 O R
pool, then split 1800 compounds; into 10 vials (180 different 42 reactions dioxolanes in each vial); add 1 of 10 acid chlorides to each vial
1800 dioxolanes + 90 diols = 1890 different small molecules from 42 reactions
S. Sternson, J. Louca, J. Wong, S. Schreiber JACS 2001, 123, 1740. Building Blocks Used in the 1,3-Dioxane Library
MeO (c) MeO NHFmoc
MeO MeO NHFmoc
(d) O O O O O O Me O S Me Cl Me NCO Cl O N Cl O S Cl O NCO O O Me
O O O
O O N C S Me Cl O O OO Me S Cl N Me OOMe NCO H O Me O Me O
Split-Pool Diversity-Oriented Synthesis of a 1,3-Dioxane Library
H H H 3 H 3 N R3 N R3 N R N R O O O O
OO OO OO OO 2 2 2 NR SR2 NR SR 1 1 1 HO R O R1 O R HF-Pyridine HO R H H 3 N R3 N R H H 3 N R3 N R O O O O OO OO 2 SR2 NR OO OO 2 NR2 1 SR O R1 O R 1 HO R1 HO R
1890 compounds; 1890 stock solutions each bead contains in 7μl of DMSO one type of molecule
place each bead in a separate well of a 384-well plate
S. Sternson, J. Louca, J. Wong, S. Schreiber JACS 2001, 123, 1740. Screening an Unbiased DOS Library Uncovers New Molecules to Explore Biology
S. Sternson, J. Louca, J. Wong, S. Schreiber JACS 2001, 123, 1740.
Binding assay: 1. Activation of Glass Slides
OH OH OH
H2 SO4 SiOO Si Si H2O2 1% SOCl2 glass ("piranha") 0.1% DMF slides ~12 h THF, 4h
i-Pr i-Pr Si R O RRR Cl Cl Cl HF•Py/THF; OOO Si O SiO Si then TMSOMe Si O SiO Si
HO-R; add DMF
P. Hergenrother; K. Depew; S. L. Schreiber, J. Am. Chem. Soc. 2000, 122, 7849-7850. Small Molecule microarraying robot Biomimetic Diversity oriented Synthesis
Combinatorial chemistry is mainly used for optimization
Me R4 O R2 O N N
R3 Cl N N HO
R1 valium-a benzodiazepin benzodiazepin-based Fmoc that binds to the GABA library design receptor O NHFmoc O NH O FmocHN O O R R2 NH R2 3 2 1. piperidine R2 NH EDCI, HOBt O O 2. O O R R1 R 1 HMP F 3 HMP O NHFmoc R1 O HO2C OLi R4 R4 R2 H O R2 O R2 O N N O N N , R4X R R R 1. piperidine 3 3 3 N Bn N TFA N HMP O O HO 2. AcOH HMP
R1 R1 R1
85-95% yield B. Bunin and J. Ellman J. Am. Chem. Soc. 1992, 114, 10997. Combinatorial chemistry library of natural products analogs
Me Me R OH OH 1 H H N Me N R N N 2 O O
N N H H R 3 O Indolactam V- Library based upon O a PKC activator indolactam O O
R1 OH OH O OBn R1 R1 CbzHN H H 1.TfO N N , HCl HN NH2 O HN 1. O O 2,6-lutidine OCO2Bn + + 2. H2, Pd/C, H 2. H2, Pd/C, H N 3. TBTU, HOBT N N H H polystyrene H 3. Cl O
O O R OH 1 H R1 O N H R2 N 1. R CHO, NaBH(OAc) , N O 2 3 R2 N DMF-AcOH O 2. I2, pyridine TFA-H2O N 3. PdCl2(PPh3)2, CuI H N R3 R3 H O 31-membered library: no improved PKC activator found R3 H. Waldmann et al. Angew. Chem. Int. Ed. Engl. 1999, 38, 2902.
Natural products as reagents to explore biology
O CH3O N Me H CH3O CH3O O
colchicine OCH3 colchicine is used to discover tubulin colchicine alters microtubule dynamics
OH H brefeldin reversibly O blocks protein HO O Me secretion by disrupting golgi structure H brefeldin A Biomimetic Diversity oriented synthesis
Amaryllidaceae Alkaloids: OH Divergent Biosyntheses and OH H R Diverse Biological Activities N RO RO N O RO RO HO crinines pretazettines (Inhibits Cell Division (antiviral) enzyme-mediated in Yeast) RO cyclizations N OH RO R OH H norbelladine HO O H R RO N H N RO RO galanthamines lycorines (acetylcholine (antimalarial) esterase inhibitor) D. H. R. Barton and T. Cohen Festschrift A. Stoll; Birkhauser, Basel, 1957.
Biomimetic Diversity oriented synthesis
Biosynthesis OH OH
HO O Synthesis of natural MeO enzymes MeO product analogs to optimize drug-like N N properties H Me norbelladine galanthamine natural selection Biomimetic Diversity-Oriented cell-based Synthesis screens OH O 4 2 Discovery of PO O galanthamine-like Br Br H H molecules with O O biological properties PO N HO N beyond those of the O bead 1 H O 3 natural product P efficient synthesis of thousands of natural protein binding product-like molecules screens Biomimetic Diversity oriented synthesis
Biosynthesis
OH O O OH P-450 like OH enzyme OH cyclize O O MeO MeO MeO MeO
NH NH NH NMe 4'-O-Methyl N-Demethyl Galanthamine norbelladine Spiro-dienone narwedine
Biomimetic solid-phase synthesis
OH O O Add Building PhI(OAc)2 Pd(PPh3)4 H Blocks O O O (CF ) CHOH Br 3 2 Br morpholine Br CH2Cl2 H O O O Si 3 O N O N HO NH H iPr iPr Alloc Alloc Narwedine Norbelladine 500μM Spiro-dienone Equivalent Equivalent polystyrene
Diversity-Generating Reactions
R OH O 1 O PPh3, n DIAD R2SH, BuLi O >80% O >80% purity Br purity Br O O Si 3 R1 Si 3 HO N O N H iPr iPr H iPr iPr H H O
R3CHO, NaCNBH 3 O or R3COCl; R2 Br S R3NCO Acylation O R Si 3 >80% purity 1 O N H iPr iPr H R4 O N
H NOR ,or O 2 4 O R H NNHSO R R Br S 2 2 2 4 Br S 2 O >80% purity O R Si 3 R Si 3 1 O N 1 O N H iPr iPr H iPr iPr R3 R3 First Step of the Galanthamine-Based Library
Reductive amination of a tyrosine-derived amine prepares a norbelladine-like starting material on the solid-phase
from tryosine OH OH O 1. CH(OCH3)3, CH2Cl2 O Br wash, then NaBH3CN, AcOH Br H iPr MeOH-THF, 23ÞC O i i O CHO H Pr O Si Pr O O N Si iPr H2N O 3 2. Cl O O Q 3 i Q Pr2EtN, CH2Cl2, 23ÞC norbelladine equivalent = 500-600 micron Q 1% DVB polystyrene OH
OH Nature’s starting MeO material for galanthamine biosynthesis NH 4'-O-Methyl norbelladine
Pelish, Westwood, Feng, Kirchhausen, Shair, J. Am. Chem. Soc. 2001, 123, 6740-6741.
Solid-Phase Biomimetic Oxidative Cyclization
The oxidant PhI(OAc)2 promotes a biomimetic oxidative intramolecular biaryl coupling reaction to generate a seven-membered ring OH O
O PhI(OAc)2, O (CF ) CHOH-CH Cl , Br i 3 2 2 2 Br H Pr 23ÞC H iPr O O Si i i O N Pr O N Si Pr
O 3 O O Q O 3 Q norbelladine equivalent Ph I O OAc
-2AcOH O -PhI Br H iPr O Si i O N Pr O O 3 Q
OH O P-450 like OH enzyme OH MeO MeO
NH NH 4'-O-Methyl norbelladine Spiro-dienone Deprotection initiates a stereoselective biomimetic cyclization
Pd-catalyzed triple deprotection yields a bisphenol which adds selectively to one of the two diastereotopic enones. The stereochemistry is controlled by a remote stereogenic center.
diastereotopic enones
prochiral carbon O O
O O Pd(PPh3)4 morpholine-THF Br i 23ÞC Br H Pr H iPr O i O O N Si Pr HO N Si iPr O H O 3 Q 3 Q what is the mechanism of this reaction? how do you account for the stereoselectivity?
O O
OH cyclize O MeO MeO
NH NH N-Demethyl Spiro-dienone narwedine
The First Diversification Reaction: A Mitsunobu Reaction
Mitsunobu coupling of the solid-phase phenol with five alcohols and a “skip”. The skip refers to a flask where no building block is added so that the phenol is represented in the library
i CO2 Pr NN O O i PrO2C R OH, PPh O 1 o 3 O THF, 0 C (2X) Br Br i H iPr H Pr O O R1 i HO N Si iPr O N Si Pr H H 3 Q 3 Q
OH OH Skip
OH NO2 OOH OH building blocks that gave >80% yield were used
i H CO2 Pr NN and Ph P=O By products: i 3 PrO2C H The Second Diversification Reaction: Conjugate Addition
A diastereoselective conjugate addition is achieved with seven thiolates and a skip so that the enone is represented in the library
O O
O O R Br R2SH, 2,6-lutidine 2 i Br S i H Pr nBuLi, THF 0ÞC H Pr R O O 1 Si i R1 i O N Pr O N Si Pr H H 3 Q 3 Q
OMe HS Skip HS O HS HS CF3 HS
building blocks that HS HS gave >80% yield OTBS
The Third Diversification Reaction: Acylation and Reductive Amination
An example of functional group diversification - introducing different types of building blocks at a single position, leading to diverse functional groups A library with diverse functionality has diverse properties: in this case amides, ureas, and amines leads to library members which are charged or uncharged at physiological pH O O
R CHO, AcOH, MeOH-THF, then NaBH CN in MeOH, 23ÞC O 3 3 O R or R COCl, 2,6-lutidine, CH Cl , 23ÞC R S 2 3 2 2 2 Br i Br S i H Pr or R3NCO, CH2Cl2, 23ÞC. H Pr O R1 i R O i O N Si Pr 1 O N Si Pr H R 3 Skip 3 Q Cl Cl 3 Q S OCN OCN O O H SMe H H
O O O building block diversity
O O O
O R O R O R S 2 2 2 Br i Br S i Br S i H Pr H Pr H Pr O R1 i R O i R O i O N Si Pr 1 O N Si Pr 1 O N Si Pr H O H H 3 EtHN Q 3 QQ3 uncharged at pH= 7.4 charged at pH= 7.4 charged at pH= 7.4 The Fourth Diversification Reaction
The ketone is transformed - with functional group diversification - into oximes, hydrazones, and a ketone (via skip) R4 O N R NH , O 4 2 R2 AcOH-MeOH-CH2Cl2, O Br S R2 iPr 23ÞC. Br S H H R O 1 Si iPr R O O N 1 O N X R3 3 Q R3 iPr iPr X = Si Q HF-pyridine, THF, 3 O O 23ÞC then TMSOMe X = H H2N N S NH2 H O NH NH2 Skip S O O OMe O NH H2N N S O H O N O 2 HO NH2 NMe2 BnO MeO NH NH2 2 building blocks that gave >80% yield
Synthesis of a 2946 Member Library
H N O NH2 O HO NH2 HS HS HS BnO O2N MeO O CF3 NH2 O O NH2 H H N S Skip Codon Skip Codon OMe O N 2 N S NH H HS HS O 2 R O O OMe 4 N H N S 2 N HS HS OTBS H O NMe2 R Br S 2 OH OR OH R1 Cl O N H S Cl R3 O NO2 O H SMe H OH Skip Codon Skip Codon O O OCN OCN OH H OOH O Synthesis of a 2946 Member Library
Library synthesized as one type of compound per 500 micron bead
Library contains one type of core structure with various appendages.
A challenge is a library with diverse core structures.
H N O NH2 O HO NH2 HS HS HS BnO O2N MeO O CF3 NH2 O O NH2 H H N S Skip Skip OMe O N 2 N S H HS O NH2 HS O R4 O OMe N H N S 2 N HS HS H OTBS O NMe2 R Br S 2 OH OR OH R1 Cl O N H S Cl R3 O NO2 O H SMe H OH Skip Skip O O OCN OCN OH H OOH O
Assay of a 2946 member library
Single beads are arrayed in each well of a 384-well plate, 18 total plates. Then, the compounds are cleaved simultaniously with HF-pyridine and re- suspended in DMSO to make 2946 stock solutions
O O H H S N S N N N O O N H S H S O O OH Br H Br H H H OH OH O N HO N O3B14 03B02 O N O N OMe H S O H O S Br H H H Br OH H OH O N O N O3K17 O 03K05 NO2
Plate 03
384 Well Plate, ~50 nmol/well, 6.7 uL DMSO/well, ~7.5 mM/well Assay of a 2946 member library
After cleavage from the solid phase, the solutions of library members are transferred using a robotic pin transfer arrayer.
Biochemical pathway
The Secretory pathway is responsible for the transport of newly synthesized proteins from the ER, through the Golgi to either the plasma membrane for secretion or to other cellular compartments
Olkkonen, V.M. et al. New Eng. J. of Med., 2000, 343, 1095. A GFP is used to screen for exocytosis
Plasma ER Golgi Membrane
Screen of the library at ~7.5mM revealed ER blockers and Golgi blockers
OH O O
O O S S Br OH Br plasma S N O N OH O membrane Golgi O
NO2 ER 14 Molecules 117 Molecules
ER Block Golgi Block Control Active compound identified
secramine O N
O Br S
OMe O N H H OH
2μM plasma golgi membrane
washing out secramine restarts trafficking- reversible inhibitor
Pelish, H.E.; Westwood, N.; Feng, Y.; Kirchhausen, T.; Shair, M. D. J. Am. Chem. Soc. 2001, 123, 6740-6741. V. Diversity Oriented Synthesis – more examples
Shikimic acid library
– DOS based upon a complexity-generating reaction (reaction-based) – Synthesis of a 2.1 million member natural product-like library – Reaction selection and building block evaluation in DOS – Quality control in DOS
R5 NH H R N 4 O O H R O N 6 H O O O Reaction Based Library
V. Diversity Oriented Synthesis – more examples
Benzopyran library
– DOS based upon privileged structure - the benzopyran core – Scaffold formation during attachment and cleavage – Directed sorting in library synthesis – The IRORI NanoKan system – Further diversification of a library after cleavage
MeO O H MeO O H O Me O Me
Privileged Structure Based Library A DOS Library Inspired by a Powerful Complexity-Generating Reaction
• A tandem esterification-[3+2] dipolar cycloaddition reaction gives a complex tricyclic structure, forming 3 bonds, 2 rings, and 3 new stereocenters in high yield and with complete stereocontrol • The efficiency of this transformation and the complexity of the product make this reaction a good candidate for the basis of a library
0.1 eq TiCl4 HO O O 4Å MS O O + N N MeO O DCE, rt, 19h 97%
O H O H O NO N H OO H H H H
O. Tamura et al. Tetrahedron 1995, 51, 107
Testing of the Reaction on the Solid Support
• The model tandem reaction was tested on beads, and proceeded successfully with modified conditions
O O O O O H HO PyBOP, NMP N OH HO MeO N N OO + rt H (SCNBu2Sn)2O >98% H2N 4Å MS, tol, rt, 12h H H H >98%
O
H2N H HN N O O H2N O2NO
PS Caproic AcidGeysen Linker Tentagel resin (cleave with hν) The shikimic acid pathway converts simple carbohydrate precursors derived from glycolysis and the pentose phosphate pathway to the aromatic amino acids (Hermann and Weaver, 1999). One of the pathway intermediates is shikimic acid, which has given its name to this whole sequence of reactions. The well-known, broad-spectrum herbicide glyphosate (available commercially as Roundup) kills plants by blocking a step in this pathway. The shikimic acid pathway is present in plants, fungi, and bacteria but is not found in animals. Animals have no way to synthesize the three aromatic amino acids—phenylalanine, tyrosine, and tryptophan—which are therefore essential nutrients in animal diets.
Chiral Template for Library Synthesis
• The starting material for the library was made from (-)-Shikimic Acid: – Source of chirality (both enantiomers were synthesized) – Provides functional groups for additional building block diversification
O O O O O HO HO Br Br OH Amberlite–IR120 OMe O OMe HO HO MeOH, 65°C CH3CN, 0°C AcO OH 18 h, 100% OH 90 min, 76% OH (-) shikimic acid methyl shikimate
O 1) NaOMe, MeOH O LiOH HO HO 0°C, 30 min OMe THF/H2O OH
2) NaOMe, MeOH O 0°C, 1 h O 50°C, 10 min, 60% 62% (–)
H. B. Wood et al. J. Am. Chem. Soc. 1990, 112, 8907 Chiral Template for Library Synthesis
• The synthesis of the (+) enantiomer of the starting material also proceeded from (-)-Shikimic Acid
O O O HO HO DEAD, PPh3 HO OH Amberlite–IR120 OMe OMe HO HO MeOH, 65°C toluene, 111°C OH O OH 18 h, 100% 90 min, 96% (–)-shikimic acid methyl shikimate
O BzOH, DEAD O LiOH HO PPh , THF BzO THF/H2O OH 3 OMe
0°C → rt, 12 h 0°C, 1 h O O 88% 59% (+)
Reaction With the Chiral Template - Scaffold Synthesis
• Synthesis of the full scaffold on the solid phase was achieved with high efficiency
OO O I O PyBOP O N H I HO HO N OH DIPEA HO N OOH + H N H2N NMP, rt, 1 h H O PyBroP, DIPEA, DMAP H >98% O (+) CH2Cl2, 0 °C → rt O O 3 x 3 h, >98%
OO O I O PyBOP O N H I HO HO N OH DIPEA HO + N OOH H N H2N NMP, rt, 1 h PyBroP, DIPEA, DMAP O H H O (-) >98% CH2Cl2, 0 °C → rt O 3 x 3 h, >98% O
S. L. Schreiber et al. J. Am. Chem. Soc. 1999, 121, 9073 Diversification Potential of the Scaffold
• The scaffold offers many potential sites for elaboration and diversification, but they must first be experimentally tested • Black arrows are transformations that were found to be experimentally viable
electrophilic capping of amine nucleophilic O addition to H I lactone N N-O bond reduction electrophilic OO H Palladium catalyzed capping of N C6 alcohol coupling reactions H H O electrophilic capping of O C5 alcohol nucleophilic addition to epoxide
Reaction Selection
• Requirements for a good library reaction:
– High yield – High purity (chemo-, diastereo-, regioselectivity) – High conversion – Solubility of reagents in solvents compatible with solid-phase synthesis – Reaction conditions compatible with solid-phase synthesis • Avoid: pressure, extreme temperature, light- and moisture- sensitivity, conditions that may break beads – Compatibility with linker used for library synthesis – If split-pool synthesis is used, the scope of the reaction is important: the reaction should not be sensitive to sterics or the presence of any functional group used in the library Examples of Reaction Evaluation
O H O H N OO N Yb(OTf)3, PhCN H N OOH H Ritter reaction: N neat, rt, 16h H H H low yield HO O O O NH O Ph Ph O O O O H PhOCOCl H Debenzylation/ N N Carbamate OOH DIPEA, DCM OOH N rt, 16h, 50% N formation: H H H H O O O O
nBu O H NH H Aminolysis N nBuNH N 2 O of the lactone: OOH O H N THF, rt. HO N H H 95% H O O O O
Diversification Sequence Development
• Attempted reactions with the amide were uniformly unsuccessful
I I O O H NH HO N nBuNH N 2 O N + OOH H O H N THF, rt, 24h HO H H N O >95% conversion H O Reversion ! O O O OH Bu3Sn (Ph3P)2PdCl2 Pd2(dba)3, AsPh3 CuI, DIPEA NMP, rt, 30h PhB(OH)2 DMF, rt, 30h Pd(PPh3)4, DMF DIPEA, rt, 30h OH NH NH O N O N O H NH HO H N HO O H O N N H H O HO O O N O H O O all reactions proceed in low yield O Diversification Reaction Sequence and Building Block Testing
• Reversal of steps solved the problem • Testing of building blocks was conducted on a representative route
H N 2 MeO MeO
I
O H O H NH OMe H NH H N N (EtCO)2O N N OOH OOH (Ph P) PdCl O O H O O H N 3 2 2 N 2-pyridinol 2,6-lutidine H CuI, DIPEA H HO N DMAP, DCM O N H H H THF, rt, 16 h 95% H O DMF, rt, 2 h O O O O O >95% O >95% O Et O
Test 50 (Ph3P)2PdCl2 Test 87 2-pyridinol Test 98 DIPC, DMAP alkynes CuI, DIPEA amines THF, rt, 16 h acids CH2Cl2, rt, 16 h DMF, rt, 2 h Pathway Development MeO R
O R H NH NH N H H N N OO H O O O N H O H H H HO N O N H R O H O O O Building Block Testing O O O Choose 30 Choose 62 Choose 62
Synthesis and Analysis of a Small Test Library
OMe CN SKIP
1 23 4 5678
O OMe OMe SKIP H N H2N 2 H2N H2NN H N H2N H2N 2 OMe 12 3 4 5 6 7 8 O O O O O O OMe O SKIP OMe HO HO HO HO HO HO HO OMe N 12 3 4 5 6 7 8 O
Atmospheric Pressure Chemical Ionization Pool 4
LC–MS analysis of 8 pools of 64 distinct compounds – 456 of 456 compounds detected Choosing An Encoding Strategy and Linker
hν (365 nm) 90 μm TentaGel photocleavage copolymer bead library chemical genetic assays compound with engineered cells photocleavable in nanowells linker
NO Ph O 2 O O O N N 50 H H Ph
Ph OO ( )m Xn OMe O binary encoding tag structure elucidation CAN oxidative by EC-GC analysis cleavage of binary code
Synthesis of the Complete Library
2 spacers 2 epoxycyclohexenol O + skip enantiomers HO O N H N H H N 2 ( ) 2 98%n N >98% H O TentaGel with 3 compounds 6 compounds Geysen linker
O H I O H R4 3 iodobenzyl N 30 alkynes N OOH OOH nitrones N + skip N H H H H >98% O 90 – 95% O O O 18 compounds 558 compounds
R5 NH R5 NH H R H R 62 amines N 4 62 acids N 4 + skip O O H + skip O O H N N HO R6 O 95 – >98% H 95 – >98% H O O O O O 35,154 compounds 2,180,106 compounds Building Blocks Used in a 2.1 Million Member Library
H N H N H N H2N H2N H N H N H NHN 2 H N H N 2 2 2 2 2 2 H2N 2 2 H2NH2N
H N O H NHN H N O O O O 2 O 2 2 H N 2 H N H N O H H N 2 H2N 2 O 2 H N H N H N H N O 2 2 O 2 O 2 2 O O H2N
MeO H N O H2N 2 N H NHN OMe N N H2N H NHN 2 2 H N Si H N Si H2N N H2NN H NN 2 2 2 OMe 2 O O 2 H2N
MeO OMe OMe O OCF3
H N H N H N H2N O H N 2 2 2 OMe 2
R H2N H2N H2N H2N 5 NH F CF3 NO2 N H R4 F Cl OMe N N C N O O H2N N H2N H2N H2N OMe H N R O O O O MeO OMe 6 NH H O O O OO H N 2 H2NOMeH2N H2N O O OO O N N F S N NH2
H2N H2N S H2N H2N N 2,180,106 compounds Me OH OH OH OH OH H O O O O O O O O O O HH HO HO HO HO HO HO HO HO HO HO MeO O O O O O O O O O O O O S HO HO HO HO N OMe HO HO HO HO HO HO O HO Cl HO
O O O O O O O O O O O O O O O O O CF3 O HO H O HO HO O O O N HO O O HO HO HO HO OMe MeO HO HO HO HO HO O O HO O O O O O O O O O O O O O OMe O O O O O CN HO HO CF3 OMe HO HO HO HO HO N HO HO N HO O HO HO HO HO MeO OMe N N CN N N CF N 3 O O OMe O O O O O O O O O O O O O O S N N HO HO HO HO O HO S HO HO HO HO HO HO OMe HO HO O Fe OMe OMe OMe
S. L. Schreiber et al. J. Am. Chem. Soc. 1999, 121, 9073
Summary of Shikimic Acid Library Development
1. Identification of a complexity generated reaction 2. Testing and adopting the reaction to the solid phase 3. Reaction pathway development 4. Building block testing 5. Quality control - Synthesis of a test library 6. Encoding Shikimic Acid Library 7. Library synthesis Reaction-Based Split-Pool Synthesis 161 Reactions, 2.1 million compounds Privileged Structure
• Privileged Structure: a structural pattern which is commonly found associated with a particular property and may contribute to that property • Example: Biological Activity – The benzodiazepine substructure is found in a wide variety of biologically active natural products
Me OH O H OH O N N O Ph Me H N HO N N N N NH2 NH H Ph O HN O Cyclopenin Anthramycin Asperlicin Phytotoxic Antitumor and Human activity antibiotic activity Cholecystokinin antagonist • Example: Asymmetric Catalysis – The binapthyl substructure is part of many effective chiral ligands
PPh2 PPh OMe 2 OH O O PPh2 OH PPh2 P O
BINAP BINOL MOP Asymmetric Enantioselective Asymmetric hydrogenation hydrosilylation hydroformylation
Benzopyran as Privileged Structure
• The benzopyran substructure is found in many diverse biologically active natural products
R1 R2
R3 O Me Me R4
OMe Me MeO O Me Me Me O H H Me O O
O O O O H HO H O O O Me OH Me Me OH deguelin daleformis electron transport ICE inhibitor Me inhibitor calanolide A HIV RT inhibitor
Nicolaou et al. JACS, 2000, 122, 9939-9967 (3 full papers) Benzopyrans: Privileged Structure?
Benzopyrans: Privileged Structure? A Plan for a Library of Benzopyrans
• Attachment to the solid phase and cyclization to the dihydrobenzopyran are accomplished simultaneously • The selenide linker is stable to strong acidic, basic and reductive conditions (and some oxidants), but is cleaved with mild hydrogen peroxide treatment • Cleavage of the compound from the solid support completes the benzopyran structure
1 1 R R R1 2 BrSe 2 - R R Br R2 Se Se 3 Me 3 3 R OH R OH Me R O Me 4 Me 4 Me Me R R R4
5 R5 R5 H O R 6 R6 Se R6 Se R diversification H2O2 7 Me R7 O Me THF, 25ÞC R7 O Me R O Me Me 8 Me R8 R8 R
Nicolaou et al. JACS, 2000, 122, 9939-9967 (3 full papers)
The First Diversification Pathway
• Addition of nucleophiles to an aldehyde scaffold was followed by acylation or Mitsunobu reaction of the resulting secondary alcohol
1 Me R Me R1 O O Me Me H 20 R2MX R2 Se Se O THF, 0ÞC OH 9 aldehydes
3 1 Cl R Me R 1 O Me R 15 Et3N, Me O 2 Me O DMAP R 2 H2O2, THF, 25ÞC R Se 3 O R O R3
O O
H 1 1 Me R Me R N O O 8 R4 Me Me R2 2 N H2O2, THF, 25ÞC R Se N Ph3P, DEAD, N CH Cl , 25ÞC 4 2 2 R R4 N N The Second Diversification Pathway
• Reductive amination followed by acylation or sulfonylation of the resulting amine
1 Me R 1 O Me R 2 O Me 20 R NH Me 2 H 2 NHR Se Se O NaCNBH3, THF-MeOH, 0ÞC
9 aldehydes 3 1 Cl R Me R 2 O R 1 15 Me R 2 Me 3 O R O N R H O , THF, 25ÞC 2 2 Me 3 Se N R Et3N, O DMAP O
1 Me R 2 O R 1 4 Me Me R 2 10 R SO2Cl N O R SO R4 H2O2, THF, 25ÞC Me 2 N Se SO R4 Et3N, 2 DMAP
The Third Diversification Pathway
• Knoevenagel condensation of a benzylic nitrile was followed by cleavage of some library members - and glycosylation of library members containing a protected phenol
1 Me R Me O 15 O Me R2 Me H NC Se Se O KOEt, THF, 25ÞC NC 9 aldehydes R2
2 If R = p-OTHPTsOH cleave 2 R = OH THF-MeOH H2O2 THF, 25ÞC Me O Me HN O O Me Me O O Se Me Me R2 CCl3 R5 NC Se 5 sugars R2
OO NC NC 2 BF3Et2O, CH2Cl2 R 4Å MS, 0ÞC OH R5 Building Blocks Used in the Benzopyran Library
Synthesis Scheme for the Entire Library The IRORI System: Kan Reactors for Solid Support
• The IRORI system carries each library member on beads contained in a plastic or ceramic capsule (“kan”) • Kans have higher loading levels than beads, and are more mechanically robust • Handling of the IRORI kans is highly automated
www.irori.com
Individual Labeling of Kans Simplifies Decoding
• Each Kan has a laser-readable tag • The Kans can be read and sorted in a high-throughput machine
• Encoding of an IRORI library: – The path of a Kan through the library synthesis is pre-determined – At each step, the Kan is sorted into the appropriate reaction vessel by a computerized sorter (2,000 Kans/hour) – At the end of the library synthesis, the path followed by each Kan is known by the computer, so no decoding of the library is necessary
www.irori.com IRORI Equipment for Synthesis of a Large Library
www.irori.com
Construction of the Library with IRORI
• Directed sorting of library members greatly simplifies the execution of a complex library synthesis plan
• Analogous to selective splitting of compounds in a split-pool library Diversification After Cleavage from the Solid Phase
• The benzopyran library could be elaborated and further diversified after cleavage from the solid phase
O O O O R2 R O OH O 2 O Se R2 OMe H Se H2O2, THF, 25ÞC piperidine, 95ÞC Me Me O O O Me 1 Me 1 Me R R R1 Me
O O O 2 R R2 2 OO O 3 R 3 O O OR O OR 3 OH Me Me R OH Ac2O OAc Amberlyst-15 acetone O Me Me Me 1 + O DMAP O Me (H ) 1 Me 1 R R R Me
Me N
DMAP = N scavenger resin
Benzopyran library
O
O N S O O
O Me Me
Benzopyran Library Privileged Structure-Based Directed Sorting 18 reactions, 10215 compounds V. Excursion: New synthetic techniques in chemistry
Microreactors in Chemical Synthesis
Changes in the way we do chemistry Chemical dimensions
Surface to volume ratio Small dimensions = short mixing time
Small dimensions = short mixing time Small dimensions = efficient heat transfer
Nu = Nusselt number; describes enhancement of heat transfer from a surface in a “real” situation if compared to conductive heat transfer.
Exact control of residence time Safety
Small volumes and better control of reaction conditions reduce risk and open new process windows (reactions at very high temp- eratures and pressures with very short reaction times)
A Closer Look at Mixing
density x velocity Reynolds number = kinematic viscosity laminar < Re = 30 < turbulent A Closer Look at Mixing
Induction of vortices (Strudel, Wirbel) by stirring, moving parts or structured channels
A Closer Look at Mixing
For all mixing techniques: Microreactor apparatus set up
Microreactor apparatus set up
Inlet Inlet 1 Inlet 2
Outlet Outlet Microreactor apparatus set up
Microreactor apparatus set up Microreactor apparatus set up
Microreactor apparatus set up Microreactor apparatus set up
Microreactor apparatus set up Microreactor apparatus set up
Lab scale reactions Michael reaction
O O
O i O O Pr2EtN, EtOH + EtO
O OEt
Synthesis of 1,3-Diketones Synthesis of Tetracyclone
o O Mixing and reaction at +80 C: O Ph Ph Base, ΔT Ph Ph + O O Butanol Ph Ph 70 - 800C Ph Ph
Mixing and reaction at + 90oC:
Aromatic Nitration
Segmented flow Landolt Reaction - Iodine Clock
- + - - - + IO3 + 6 H + 3 HSO3 ———> I + 3 HSO4 + 6 H
- + - IO3 + 6 H + 6 I ———> 3 I2 + 3 H2O
Landolt Reaction - Iodine Clock
- + - - - + IO3 + 6 H + 3 HSO3 ———> I + 3 HSO4 + 6 H
- + - IO3 + 6 H + 6 I ———> 3 I2 + 3 H2O Segmented flow
Segmented flow
Controlled precipitation In a bubble tube Segmented flow
Micro-encapsulation of drugs in polymeric microspheres
Polylactic acid; polylactic-polyglycolic acid
Lab process for microsphere preparation:
Segmented flow
Micro-encapsulation of drugs Micro mixer process Resorchin Azocoupling reaction
HO HO N 2 [Base NEt ] 3 N + BF4 N NO 2 OH OH NO2
C6H6O2 C6H4BF4N3O2 C12H9N3O4
low base concentration
high base concentration
Peptide Synthesis
(4{N-[1-(4,4-dimethyl-2,6-dioxocyclohexylidene)-3-methylbutyl]-amino} benzyl alcohol) = Dmab-OH Peptide Synthesis
Suzuki coupling Photo reactions
Combinatorial compound library synthesis
Domino reaction: Knoevenagel condensation and Hetero-Diels-Alder Combinatorial compound library synthesis
Amide formation and Knorr-Pyrrol Synthesis
Scale up
“Got a few problems going from lab scale up to full commercial scale” Asymmetric synthesis via organoboranes
Asymmetric synthesis via organoboranes
Synthesis of the chiral allylboranes Asymmetric synthesis via organoboranes
Synthesis of chiral allylalcoholes
Asymmetric synthesis via organoboranes
Synthesis of chiral allylalcoholes Asymmetric synthesis via organoboranes
Azo pigment synthesis Azo pigment synthesis
Azo pigment synthesis
10 t per year Azo pigment synthesis
Advantages:
• Constant product properties compared to batch synthesis • Short development time (18 months) from lab to plant • Switching between products is fast • Low operating cost
Summary
Microreactor systems allow a better control of reaction conditions and therefore provide advantages for • fast reactions • very exothermic reactions • handling of dangerous reaction intermediates • reactions at very high temperatures and pressures • sequential reactions
A better control of reaction conditions may reduce side reactions.
Optimization of reaction conditions is facilitated and the use of otherwise difficult accessible “process windows” becomes possible.
Simple scale up of a reaction by “numbering up” (parallel use of several microreactors)
Production of t/year in a laboratory environment Customized production on demand VI. Complexity Generating Reactions
Molecular Complexity
Defined by Our Intuition – Complexity is defined in part by the frontiers of chemistry
– Complexity resists definition, but we know it when we see it
• Molecular Size • Element and Functional Group content • Cyclic Connectivity • Stereocenter Content • Chemical Reactivity • Structural Instability
Intuitive Definition of Complexity
• Contributors to Complexity – Molecular Size Palytoxin
• 115 carbon chain • 71 stereogenic elements • Requires 42 hydroxyl protecting groups! Intuitive Definition of Complexity
• Contributors to Complexity – Element and Functional Group Content Vancomycin
Aminodisaccharide Atropisomerism Benzylic β-hydroxyls
Free Acid
Carboxamide Phenols
Intuitive Definition of Complexity
• Contributors to Complexity – Cyclic Connectivity – Stereocenter Content Ginkgolide B
• 6 fused 5-membered rings • Dense functionality • 13 of 14 ring carbons are asymmetric Intuitive Definition of Complexity
• Contributors to Complexity – Chemical Reactivity: Neocarzinostatin Chromophore
OH R1 OH HS O O H3C O H C O O 3 SR1 O O O O O O OCH3 OH HB OCH3 RO RO • •
R = H OH R = O MeHN CH H C O 3 3 1 O HO SR OH O O O OCH3 RO OH • Aglycon decomposes in 1-2 hours DNA-damaging agent
Intuitive Definition of Complexity
• Contributors to Complexity – Structural Instability • Example: Phomoidride B (CP-263,114)
Highly epimerization-sensitive O Sensitive pseudoester H O O O O O
O HO2C Reactive maleic Free Acid anhydride A Computational Definition of Complexity?
• Bertz, S. H. J. Am. Chem. Soc. 1981, 103, 3599-3601. • Using graph theory: Complexity (C) is related to the number of times (n) a given pattern can be mapped onto the molecule
The number Symmetry of patterns reduces the complexity C(n) = 2n log2 n - Σni log2 ni i
pattern
Atoms 8.0015.51 8.00 16.00
Bonds 8.0015.51 15.51 9.51
Propane 8.00 15.51 43.02 4.00 Adjacent 8.00 15.51 43.02 16.00 Bonds
• These methods are limited to simple connectivity, double bonds, etc. Influence of stereochemistry, heteroatoms, functional groups, reactivity, and stability are not effectively included
Why Complexity in DOS?
• Intuition and the desire to emulate nature – Nature makes molecules of a wide range of complexity for a wide range of functions
NH O Me N H2N N O Me HO H O N N O Me OH H N N H O Kramerixin Distamycin A Potent, broad-spectrum Antibiotic by binding to the N antifungal agent minor groove of DNA H H
S Me O Me O H N N N N OH H2N N Me H H N NH2 H2N Cl O H Me N H H OH O OH Palau'amine Epothilone B Potent immunosuppressant Anti-mitotic by stabilization by unknown mechanism of microtubules Challenges of Complexity in DOS
• Length of the Synthetic Route – Target-oriented synthesis of a complex natural product often requires 20-40 steps
Corey et al. - 31 linear steps JACS, 1988, 110, 649.
Crimmins et al. - 25 linear steps JACS, 1999, 121, 10249. JACS, 2000, 122, 8453. Ginkgolide B
– The length of a synthetic route on the solid phase is limited to 5-15 steps by purity considerations
Avg. transformation % of desired B on 10 steps ? efficiency bead 80% 11% AB90% 35% 95% 60%
Challenges of Complexity in DOS
• Planning a complex library requires reliable reactions – Testing of reactions and building blocks is conducted in a “representative” route, but quality control of all reactions in a library synthesis is impractical
– Density of functional groups and steric crowding can cause unpredictable reactivity
• A complex library requires rapid generation of complexity to minimize the number of steps Complexity-Generating Reactions
• Efficient and reliable complexity-generating reactions (CGRs) are necessary in DOS to assemble natural product-like molecular frameworks in few steps
• Reactions which rapidly create rings, stereocenters, carbon- carbon bonds, and functional groups
– Cycloaddition reactions
– Multicomponent couplings
– Tandem reactions - Two or more transformations conducted in sequence in one reaction vessel
Types of Tandem Reactions
• Tandem cascade reactions - no isolable intermediates
[4+2] [4+2] CO Me CO Me 2 + 2 MeO CCOMe CO2Me 2 2 CO2Me
• Tandem consecutive reactions - a change in conditions starts the subsequent transformations
OH OO KH, heat hυ, heat H 18-crown-6 H H Schreiber, S.L.; Santini, C. J. Am. Chem. Soc. 1984, 106, 4038. • Tandem sequential reactions - requires addition of another component
CO2Me CO2Me MeO2C TMSO
[4+2] [4+2] O Denmark, S. E.; Thorarensen, A. Chem. Rev. 1996, 96, 137-165. Design of Tandem Reactions
Thermodynamics - Keep in mind that reactions must be energetically favorable – The Benson additivity rules are useful for the quick calculation of the bond and ring strain energies gained and lost in a reaction
– The introduction of strain into a molecule is often an effective way of promoting rearrangements Approximate Selected Energies (ΔH only)
σ-bonds π-bonds rings
H-Csp3 100 C-C 85 C-C 67 3-C 28 H-Csp2 103 O-C 84 C-O 88 4-C 26 H-Csp 125 N-C 79 C-N 75 epox. 28 H-O 102 Cl-C 82 H-O2C 112 Br-C 70 H-N 103 I-C 54
Design of Tandem Reactions
Kinetics – Intramolecular reactions are greatly accelerated over an analogous intermolecular reaction
– Proximity can be used to enforce the desired reactivity
– Irreversible termination steps can be included if the product would otherwise be a small (uphill) component of an equilibrium Design of Tandem Reactions
• Key elements – Recognition of new reactive patterns in the products of a reaction
- O O O O O OMe OMe base Br Br OMe Keying Elements: O • Enolate • Leaving group
• “Programmed” starting materials – Certain functional group patterns lead to cascade reactions – e. g. spaced olefination: Heck, radical, cation cascades
I I radical initiation
initiator + spaced radical acceptors
Complexity-Generating Reactions in DOS
• Tsuge reaction followed by a [2,3]-dipolar nitrile oxide cycloaddition
O O O 1 N N N R O O H H H - N Cl N H H O O O N O O Et N 3 R1
2 R 2 N R H N O H Cl N O O OH H O N H 2 O 3 N R H H R NH2 R3HN N H H H H H O OON O N O R1 R1
Brooking, P.; Crawshaw, M.; Hird, N. W.; Jones, C.; MacLachlan, W. S.; Readshaw, S. A.; Wilding, S. Synthesis 1999, 1986-1992. Complexity-Generating Reactions in DOS
• Tandem esterification - nitrone [3+2] cycloaddition approach to a tetracyclic scaffold
O PyBroP O N X HO O O or HATU N + O O H N O HO ()0,1 DMAP X i N O Pr2NEt H O
() O H 0,1 N [3+2] OOH N HH O O
Tan, D. S.; Foley, M. A.; Shair, M. D.; Schreiber, S. L. J. Am. Chem. Soc. 1998, 120, 8565- 8566.
Complexity-Generating Reactions in DOS
• A three-component condensation gives tetrahydroquinolines from simple starting materials
OO OO
+ + TFA N NH2 CHO
OO OO
H H
N HN H H
Kiselyov, A.S.; Armstrong R.W. Tetrahedron Lett. 1997, 38, 6163. Complexity-Generating Reactions in DOS
• An oxidative coupling - hetero-Diels-Alder approach to Carpanone-like molecules
Me Me Me Me Me Me H OH Diels- O O O PdCl O O + 2 Alder H O O OH O O O 46% O O O O O O Carpanone
Chapman, O. L.; Engel, M. R.; Springer, J. P.; Clardy, J. C. J. Am. Chem. Soc. 1971, 93, 6696.
R1 R4 R4 1 4 R R1 R O Diels- HO O + PhI(OAc)2 Alder H OR 2 O O 3 R R2 O H O R2 HO O R3O OR3
Lindsley, C. W.; Chan, L. K.; Goess, B. C.; Joseph, R.; Shair, M. D. J. Am. Chem. Soc. 2000, 122, 422-423.
Complexity-Generating Reactions in DOS
• A biomimetic oxidative phenolic coupling used to make a library of Galanthamine-like molecules
OH OH
OH O MeO enzymes MeO
N H NH Norbelladine Galanthamine
Eichhorn, J.; Takada, T.; Kita, Y.; Zenk, M. H. Phytochemistry 1998, 49, 1037-1047.
OH O O O O Br Br O PhI(OAc)2 Pd(PPh ) O 3 4 Br O morpholine O O N O N HO NH O O O O
(a) Pelish, H. E.; Westwood, N. J.; Feng, Y.; Kirchhausen, T.; Shair, M. D. J. Am. Chem. Soc. 2001, 123, 6740-6741. (b) Kita, Y.; Arisawa, M.; Gyoten, M.; Nakajima, M.; Hamada, R.; Tohma, H.; Takada, T. J. Org. Chem. 1998, 63, 6625-6633. Complexity-Generating Reactions in DOS
• A sequential 1,4 cuprate addition - enolate alkylation approach to a library based on Prostaglandins
1) Bu3Sn O OTMS OTBS Li2Cu(CN)Me2 O O OO O O 2) TMSCl OTBS
O MeLi; CO2Me
TfO CO Me O 2 OO OTBS
Chen, S.; Janda, K. D. J. Am. Chem. Soc. 1997, 119, 8724-8725.
Complexity-Generating Reactions in DOS
• A tandem Knoevenagel-Ene reaction gives complex cyclic compounds
R O R O ( )n O O O ( )n MeO O MeO O
R O
O ( )n
MeO O
Tietze, L.F. Angew. Chem. Int. Ed. 1996, 35, 651. Complexity-Generating Reactions in DOS
• An Ugi - Diels-Alder sequence gives a complex tricyclic core from four simple components
Ugi 3-Component Coupling HN O OHC O CN , O H2N N OR O OR H HO C N 2 HN Br O O Br
HN O H Diels- O N Alder OR
H O H Si O HN R= O Br
Paulvannan, K. Tetrahedron Lett. 1999, 40, 1851-1854. Lee, D.; Sello, J. K.; Schreiber, S. L. Org. Lett. 2000, 2, 709-712.
Complexity-Generating Reactions in DOS
• The tricyclic core is subjected to an olefin metathesis cascade to give a rearranged tetracyclic core
O O N HN H H O O N OR N OR Br O Br O H H H H KHMDS N HN O O Br
N N O H OH 1) Cl Ru N N Cl Ph O PCy3 H O O 2) HF•py H H Br N
Lee, D.; Sello, J. K.; Schreiber, S. L. Org. Lett. 2000, 2, 709-712. Tandem Cycloadditions and Sigmatropic Rearrangements
Tandem Cycloadditions and Sigmatropic Rearrangements
Radical cyclization
Heck Cascades
• Alkyne or 1,1-dialkylalkene groups are positioned three to five carbon apart leading to the formation of 5,6, and 7-membered rings
Zipper-mode Spiro-mode
I I SiMe3 SiMe3
Dumbbell-mode Linear fused-mode
SiMe3 SiMe3 I I Tandem Heck Reaction
• Intermolecular Heck reaction followed by intramolecular one • Syn-Carbopalladation leads to only one geometric isomer
Stable Organo-Pd intermediate without syn β-H
3 mol %
Pd(PPh3)4 CO2Me I 2.5 eq. Et N 3 L Pd 100 oC, 12 h n Me Me Me Me LnPd 80 % MeO2C
Syn-carbopalladation
E. Negish. et al. J. Org. Chem. 1989, 54, 2507
Tandem Heck Reaction
• Intermolecular Heck reaction terminated with intramolecular one R OH
Pd2(dba)3-CHCl3 30 % PPh3,Et3N H
toluene LnPd 52 % H TBSO OTBS Br TBSO OTBS
R R
OH H H H
PdLn PdL H n TBSO OTBS TBSO OTBS TBSO OTBS
B. M. Trost et al. J. Am. Chem. Soc 1992, 114, 9836 Sequential Tandem Heck Reaction
• Two Heck reactions are achieved in one-pot
OtBu (R: o-tol) Syn- carbopalladation 10 % R R Pd(OAc) , OtBu P OtBu 2 Pd OAc L Pd H 22 % PPh3, n 2.0 eq Br H H Br Br 60 oC, 60 h. H 120 oC H MeO CH3CN, DMF MeO nBu N(OAc) More 4 Reactive Syn-Elimination He OtBu Ha He OtBu LnPd Ha L Pd n H H H H H H MeO MeO 63 %
L. Tietze et al. J. Am. Chem. Soc. 1998, 120, 8971
Stable Neopentyl Organo-Pd-intermediate
• Strained tricyclic system
10 % Neopentyl-type Pd(OAc)2, 20 % LnPd A PPh , 3 R PdLn R
H H Ag2CO3, R THF, reflux OTBS IOTBS OTBS
Me O PdLn R R slow H H H H cyclization Pd(0) HO2C H OBz OTBS OTBS HO > 90 % Scopadulic acid
L. E. Overman et al J. Am. Chem. Soc. 1999, 121, 5467 Chemistry of Stable Organo-Pd Intermediate
An Opportunity for Diversification
R R R2 R1 2 1 R Nu R R3 Heck 3
Nu R
R2 R1 PdLn R3 Stille Coupling CO
RSnR'3 R1 R2 RB(OH)2 R R2 R1 O R3 Suzuki Coupling R3 PdLn
R2 R1 R R3
Sequential Tandem Heck-Suzuki Reaction
• Three component coupling reaction on solid support • Stoichiometric amount of Pd used X = 4-MeO R = 4-MeOC6H4
= 4-Me = 4-MeC6H4 = H
TMS TMS TMS
OO OO OO Pd(PPh3)4 RB(OH)2 N N X N X ArBr, THF PPh3, Na2CO3 66 oC PdLn THF, 66 oC R H H O O O without available syn-β-hygrogen
J. A. Ellman. et al J. Org. Chem. 1996, 61, 4494 Heck-Carbonylation and intermolecular Nucleophilic addition
• Three component coupling reaction
PdLn L Pd n PdLn I Pd(0)
CO CONH N N 2 N N H SO2Ph SO2Ph SO2Ph SO2Ph N H2N R O η3-allyl-Pd
CO CONH2 O O PdL Pd(0) H n' N PdLn' N R H O NH2 N N R N SO2Ph SO Ph 2 SO2Ph
F. Vöglte. et al “Stimulating concepts in Chemistry” pp 56
Multicomponent Reactions
• Multicomponent reaction: A reaction in which three or more reagents react in one pot to generate a product containing atoms from all reagents.
CO2H CO2H CHO CaCO Mannich Reaction: 3 H2N O N O CHO H2O, 25 ºC CO2H CO2H O O O 0.1 CpRu+ O O H O AcO 0.15 CeCl 3 H DMF, 60 ºC benzene O 80% OAc 80 ºC, 90% H O O OAc Interrupted three component reaction Ru catalyzed coupling followed by Diels-Alder
R. Robinson J. Chem. Soc. 1917, 111, 762. B. Trost and A. Pinkerton JACS 1999, 121, 4068. Advantages of Multicomponent Reactions
• Complexity is generated through the combination of multiple functional groups. • Diversity is easily incorporated in one step by varying the components. • Deprotection steps are often avoided. • One pot reactions are ideal for automation. • Efficient solid phase or solution phase synthesis is possible.
• Disadvantage: Generally cannot do split-pool synthesis
Two Component vs. Multicomponent Synthesis
• Three component synthesis can make 8 products in 4 fewer reactions than parallel two component synthesis. Parallel Two Component Couplings Three Component Couplings ACE ACE E C, E
AC C, F C ACF ACF F A A E ADE ADE D, E D AD ADF ADF F D, F
BCE C, E BCE E BC C, F C BCF BCF F B B E BDE BDE D BD D, E BDF BDF F D, F 12 reactions 8 reactions Multicomponent vs. Split and Pool Synthesis
• Split-pool two component synthesis can make 8 products in 4 fewer reactions than three component synthesis.
Three Component Couplings Split-Pool Two Component Couplings
C, E ACE ACE
C, F ACF BCE
A AC ADE ADE D, E pool A C pool E ADF BC BDE D, F
BCE B pool ACF C, E D pool AD F
C, F BCF BCF B BD BDE ADF D, E
BDF BDF D, F 8 reactions 4 reactions
Multicomponent vs. Split and Pool Synthesis
• Parallel multicomponent synthesis: – Requires more steps – Does not require tagging – Often avoids deprotection steps – More easily automated
• Interrupted multicomponent reactions potentially could be used for split and pool synthesis. The Multicomponent Ugi Reaction
• In the Ugi reaction, an aldehyde, an amine, a carboxylic acid, and an isocyanide couple to form an α-amino acylamide.
R2 O O O 2 4 R3 N R4 H2N R C N R 1 3 N R H R OH H O R1 – Reactions usually are run in methanol or ethanol at high reagent concentrations (0.5 to 2 M). – Exothermic, ice bath cooling is often required. – Precondensation of the amine and aldehyde can improve yield. – Lewis acids can accelerate the reaction.
A Domling and I. Ugi ACIEE 2000, 39, 3169.
Preparation of Isocyanides
• Only 12-15 isocyanides are commercially available. • Isocyanides can be prepared by dehydration of a formamide or derivatization of another isocyanide.
ClCO2CCl3
NEt3, CH2Cl2 O 0 ºC to 25 ºC 75-98% R N H OR: RNC H POCl R = 1º, 2º, or 3º alkyl, 3 benzyl, aryl, etc. HN(iPr)2, CH2Cl2 0 ºC to 25 ºC 53-89%
Li R NC R-X NC NC RX = MeI, BnBr, or CyBr
G. Skorna and I. Ugi ACIEE 1977, 16, 259. R. Obrecht, R. Herrmann, and I. Ugi Synthesis 1985, 400. P. Tempest, S. Brown, and R. Armstrong ACIEE 1996, 35, 640. Isocyanide Chemistry
• The isocyanide reacts carbene-like by the α-addition of both a nucleophile and electrophile. electrophilic
R4 NC R4 NC
nucleophilic
Nu-, E+ Nu R4 NC E
A Dömling and I. Ugi ACIEE 2000, 39, 3169.
Mechanism of the Ugi Reaction
• The coupling occurs by isocyanide trapping of a reversibly formed iminium and carboxylate, followed by an acyl migration. O H 3 HO R N R1 O H R1 1 2 2 NH2 2 N R R R R OR3 O 4 R4 NC R NC
O 2 R R O H 4 3 N O R R3 N R4 2 N O H R N R4 N 2 H 1 R N O R1 R OR3 R1
A Dömling and I. Ugi ACIEE 2000, 39, 3169. Ugi Reactions in TOS
• The Ugi reaction gives more than twice the yield of the dipeptide fragment than does linear peptide coupling. • Analog synthesis is also simplified.
CO Me MeOH CO Me O 2 NH2 hexanes 2 25 ºC N ZHN CO2H CN ZHN N H H CHO 59% O OBn OBn O CO H 2 O N N H O NH H N NH O
O CO2H motuporin subnanomolar protein phosphatase inhibitor
S. Bauer and R. Armstrong JACS 1999, 121, 6355.
Stereoselective Ugi Reaction
•ZnCl2 forms a rigid chelate with the imine to control the stereoselectivity of the Ugi reaction.
AcO AcO ZnCl2 AcO O AcO O O NH2 OHC O N Ac NAc MeOH Ac HN ZnCl2 -75 ºC O
CO2H CN AcO O O O AcOO N N 85% Ac NHAc >99% ds H
I. Ugi et al. ACIEE 1995, 34, 1104. Ugi Reaction in DOS
• Ugi Reactions are highly utilized for DOS:
– High generation of diversity • All four components can be varied. • Components are commercially available. • Reaction variations access diverse structures
– Compatible with large library size • The reaction is amenable to automated parallel synthesis. • Reactions can be run in solution or on the solid phase.
Solid Phase Ugi Reaction
• A library of 96 sialyl Lewis x mimetics was synthesized on the solid phase by a four component Ugi reaction.
HO HO OH OH O OH OH CN CO2Me 1. CH2Cl2, THF, O O 5 eq. MeOH, 25 ºC 2.5 eq. CHO O N CO2Me 2. 20% TFA H H2N in CH2Cl2 N CONH2 N 50%, high purity HO2C CO2H H 1 eq. O HO2C 1 eq. HO OH OH OH O HO HO2C HO NHAc HO O O O OO OR AcHN HO O HO OH OH sialyl Lewis x
R. Armstrong, et al. JOC 1996, 61, 8350. Fluorous Phase Ugi Reaction
• Fluorous tagged Ugi products can be isolated from excess reagents by fluorous phase extraction. O O
OH H 1. CF3CH2OH (Rfh)3Si 90 ºC organic phase Rfh = CH2CH2C10F21 17 eq. (contains reagents) 2. extraction NC fluorous phase NH2 with FC-72 (fluorocarbon) (contains product) 3. wash with 17 eq. 17 eq. benzene
O TBAF O H H N THF, 25 ºC N N N 84% O O (Rfh)3Si >95% purity
D. Curran, et al. JOC 1997, 62, 2917.
Ugi Variation: Bifunctional Reagents
• Linking two of the reaction components results in the formation of cyclic products.
H O N MeOH NH2 CO2H CO 25 ºC 2 O H N N H CN CO Et 2 EtO2C N 85% EtO2C N
O O
O
C. Hanusch-Kompa and I. Ugi Tetrahedron Lett. 1998, 39, 2725. Ugi Variation: Cyclization Strategies
• Ugi products can be converted to a diverse range of structures using various cyclization strategies. O R3 SR4 R1 N R2 O 5 4 RSH R R 1 AcCl R CO2H R1 R3 2 3 N R NH2 O R Ugi H R2 3 1 N O R CHO R N 2 2 R 1 NC R O HO N R RO O RO O O R2 N 3 R R N H O T. Keating and R. Armstrong JACS 1996, 118, 2574.
Ugi Variation: Cyclization Strategies
• An Ugi, ring-closing metathesis sequence can be used to make β-turn mimetics.
H N NHBoc CH2Cl2, MeOH 25 ºC HO2C O NH 2 CHO CN CO2tBu
O O NHBoc NH .TFA HN HN 2 1. Cl2(PCy3)2Ru=CHPh DCE, 80 ºC O O O O 2. TFA, CH2Cl2 N 25 ºC N N CO2tBu N CO2H H 55% H
A. Piscopio, J. Miller, and K. Koch Tetrahedron 1999, 55, 8189. Passerini, Wittig
• The Passerini three component reaction is like an Ugi reaction but without the amine.
OH HO C HO C 2 2 O O LiBr, NEt3 H THF N (Et2O)OP (Et2O)OP O NC tBuO C tBuO2C OH 2 O OH OH O PO(OEt)2 O O N O H H N O N 81% O tBuO C 2 O O O tBuO2C tBuO2C (EtO) OP 2 OH OH OH
A. Dömling, et al. Org. Lett. 2001, 3, 2875.
Multicomponent Cycloadditions
• A dienophile traps the reversibly formed diene for a three component Diels Alder reaction. O O O Ac2O H cat. p-TSA NH NH2 NMP, 120 ºC O
O
NH NHNOO O NH O O NH [4+2] O
M. Beller, et al. Org. Lett. 2001, 3, 2895. VII. Chemical Diversity
Hypothesis Based Research
Hydrogen bond Problem: Find a molecule capacity that can block protein trafficking. solution Only hydrophobicity, polarity and x hydrogen bonding capacity have 7 5 an influence on the molecule’s
ability to block protein trafficking x 6 Polarity
y it c 3 2
obi x oph x dr molecule chosen y H based on the initi 4 1 hypothesis
- Success depends on the quality of the hypothesis - Enormous successes have been achieved using this approach - As the complexity of the problem increases our ability to make the initial hypothesis and to use results to make new hypotheses diminishes Diversity Based Research
Hypothesis Hydrogen bond Diversity Hydrogen bond Based capacity Based capacity 8 17 14 x 7 solution solution x 7 5 5 16 11
6 x 15 Polarity 6 12 18 Polarity 9 3 2
13 3 2 x
x molecule chosen based on the initial 4 1 hypothesis 4 10 1
- Diversity based research - Hypothesis based research - Success depends mostly on the - Success depends on the quality of diversity of the library that is the hypothesis, which in turn depends screened for a solution on the information available about the Diversity is a measure of how problem and the complexity of the well the available space is problem covered and is independent of the complexity of a problem - Many problems in chemistry and biology are multi-variable problems for which it is difficult to make an accurate (productive) initial hypothesis - Introducing a hypothesis into diversity based search (e.g. privileged structure) can significantly reduce the dimensionality or size of the space that should be covered
Diversity
O diastereoisomers CN EtO O N O O N N HO O EtO O HO N HO N N
O HO S N O EtO O N HO N enantiomers HO Building block Stereochemical diversity diversity
H O O N Me NH Me N Tos N
O O O S Br Br S Br S
OH O N O N OH O N OH O O HN Functional group diversity
Molecular framework diversity Diversification Strategies
• The number of diversity positions used contributes to the total number of compounds in the library more than the number of building blocks used to diversify each of them
200 building ABblocks at each diversity position 40,000 compounds (2002)
A 6 building F blocks at each diversity position E B 46,656 compounds (66) C D
Building Block Selection
• Tools for building block selection – Databases of commercially available compounds that allow substructure search, e.g. find all primary alcohols – Computer programs that choose a subset of n compounds from a set of commercially available ones, retaining the diversity of the original set • Desirable properties of building blocks – Commercially available • Building blocks should be introduced using reactions that require the presence of a common functional group – Compatible with the synthesis plan • A building block must contain only functional groups that are compatible with reactions that will be used after the building block is introduced • Order in which diversity positions are elaborated can alleviate some of the constraints Building Block Selection
• Diverse physical and chemical properties – hydrophobicity and hydrophilicity + I OMe NMe I I I 3
hydrophobic hydrophilic – Hydrogen bond donors and acceptors O OH NH O R R H R HRRRN N O H H acceptor donor accepor acceptor and donor and donor – Acidic and basic groups O O NH2 R H R R N R O H acidic basic neutral –Size HO Me HO HO HO
Building Block Selection
• Biasing elements – metal binding elements
R R HO R 1 2 R1 R2 OH 1 N Ph3PPPh3 H2NNH2 R2 R3 – reactive groups • nucleophiles
R N RNH2 RSH N
• electrophiles O O R1 R R Functional Group Diversity
• Functional groups can be a source of diversity
OMe O H Functional group diversity elements: N N S H O O O O N Ar N R O S N S Br O OO H S R R RR OH Br RR H O N OH sulfonyl oxime ketone O O N hydrazone N O OMe N O R O R R N R N N R R RN O H H H S Br H urea amine amide OH O N
Diversity Potential of a Functional Group
• TOS - One functional group is not a priori superior to any other • DOS - Diversity potential of a functional group is a part of a library design
OR3 R 3 R2 O R2 ORO N 1 R R1 OR4 S 3 R O Ar R N 2 O 1 OH R2 O R R 1 3 O R O OH R3 O 2 R1 R3 NHR3 R1 O R H R1 HN 1 O O O O R R2 R1 3 R O O R1 N 1 R H OR R H 1 2 O 4 NNH O NS R3 HN R2 R3 R1 R R O 2 R OH R 1 1 1 R1 O OR2 All transformations are achieved in one step Stereochemical Diversity
Stereochemistry can be used as a source of diversity
O diastereoisomers Stereochemical diversity EtO O elements: N - stereogenic center - plane of chirality HO - axis of chirality O - double bond EtO O N
HO
O EtO O N
HO enantiomers
Why is Stereochemical Diversity Important?
Different stereoisomers can have dramatically different properties. Examples from asymmetric catalysis and chemical biology:
Thalidomide
O O O H N 1. catalyst F3C N H 2. TFAA NO NO H + HCN CN N N O OH O OH
S S S-thalidomide: teratogen, R-thalidomide: safe anti- H H Ph N Ph N causes severe birth defects nausea agent N N N N H H H H O N O N HO This compound was originally 10% ee HO 95% ee
t administered as a racemic mixture tBu OMe Bu OMe Stereochemical Diversity: TOS vs. DOS
• Palytoxin has 64 stereogenic centers. 2n stereoisomers are possible, where n is the number of stereogenic centers. For palytoxin there are 1.85X1019 possible stereoisomers!
TOS: The goal is to synthesize one stereoisomer. Kishi accomplished a synthesis of one stereoisomer of palytoxin: Kishi et al. J. Am. Chem. Soc. 1994, 116, 11205.
Stereochemical Diversity: TOS vs. DOS
• Palytoxin has 64 stereogenic centers. 2n stereoisomers are possible, where n is the number of stereogenic centers. For palytoxin there are 1.85X1019 possible stereoisomers!
DOS: The goal could be to synthesize all stereoisomers in one synthesis, but not as a mixture of stereoisomers. In split-pool synthesis, each bead must contain one stereoisomer. Synthesis of L-Hexoses
• Challenge: Develop one synthesis route that provides access to all stereoisomers of the hexoses • This could be an example of DOS
CHO CHO CHO CHO HO HO HO HO OH OH OH OH HO HO HO HO OH OH OH OH HO HO HO HO L-allose L-altrose L-mannose L-glucose Common Starting Material CHO CHO CHO CHO HO HO HO HO OH OH OH OH HO HO HO HO OH OH OH OH HO HO HO HO L-gulose L-idose L-talose L-galactose
S.Masamune, K.B. Sharpless et al. Science 1983, 220, 949
Total Synthesis of L-Hexoses
• Sharpless epoxidation is used to establish the stereochemistry of the two stereogenic centers • The Payne rearrangement allows functionalization of the terminal carbon atom
Ti (OiPr)4 (+)-DIPT Ph O O PhSH, NaOH OH RO t-BuOOH OH Ph H2O - t-BuOH DCM R=CHPH2 heat 92% 71%
PhSH, NaOH - H O - t-BuOH SPh 2 OH OH O heat RO RO RO OH SPh Payne O OH rearrangement
S.Masamune, K.B. Sharpless et al. Science 1983, 220, 949 Total Synthesis of L-Hexoses
• The aldehyde was generated from the sulfoxide by a Pummerer rearangement
RO RO MeO OMe OH mCPBA + Ph RO SPh S SPh O O POCl (cat.) O O O- OH 3 DCM, -78°C
- - OAc OAc RO RO RO OAc H NaOAc Ph + Ph + Ph O S S O S O 93% O Ac2O O O O over 3 steps heat Ac Pummerer rearrangement
Total Synthesis of L-Hexoses
• The stereochemistry at C-4 was diversified by establishing two pathways for hemithioacetal hydrolysis, only one of which provides an opportunity for epimerization to the thermodynamically favored configuration at C-4 RO OAc Ph O S O
DIBAL-H K2CO3, DCM, -78°C MeOH, rt RO RO O- O- Ph Ph O S O S O O
RO RO RO RO O O O- O
O H O H O H O H O O O O
no epimerization thermodynamic under the reaction product conditions Total Synthesis of L-Hexoses
RO RO O O
O H O H O O
1. Ph3PCHCHO 1. Ph3PCHCHO (E:Z>20:1) RO RO (E:Z>20:1) 2. NaBH, MeOH 2. NaBH, MeOH
O OH O OH O O
SAE, (+) DIPT SAE, (-) DIPT SAE, (+) DIPT SAE, (-) DIPT 76% 84% 76% 73% RO RO RO RO OOOO O OH O OH O OH O OH O O O O
1. NaOH, PhSH 1. NaOH, PhSH 1. NaOH, PhSH 1. NaOH, PhSH H2O - t-BuOH H2O - t-BuOH H2O - t-BuOH H2O - t-BuOH 2. 2,2 methoxy 2. 2,2 methoxy 2. 2,2 methoxy 2. 2,2 methoxy propane, POCl3 propane, POCl3 propane, POCl3 propane, POCl3
RO RO RO RO O O O O O O O O O O O O O O O O SPh SPh SPh SPh
Example of Reagent-Based Stereocontrol in DOS
RO RO O O RO OH O H O H O O
RO RO RO RO O O O O O O O O O O O O O O O O SPh SPh SPh SPh
AAAAB BBB O O O O O O O O CHO CHO CHO CHO CHO CHO CHO CHO O O O O O O O O O O O O O O O O
RO O RO O RO O RO O RO O RO O RO O RO O CC C CC CCC
CHO CHO CHO CHO CHO CHO CHO CHO HO HO HO HO HO HO HO HO OH OH OH OH OH OH OH OH HO HO HO HO HO HO HO HO OH OH OH OH OH OH OH OH HO HO HO HO HO HO HO HO L-allose L-altrose L-mannose L-glucose L-gulose L-idose L-talose L-galactose
A =1. mCPBA B =1. mCPBA C = 1. TFA - H2O 2. AcOH, NaOAc 2. AcOH, NaOAc 2. H2, Pd-C 3. DIBAL-H 3. NaOMe, MeOH Molecular Framework Diversity
• Molecular framework (MF) is the largest structural element present in all (or a subset of all) the members of the library
O Molecular frameworks CN Me N O O O N EtO O O N EtO O Br S N HO N HO HO O N OH O O Me N H O O O N O EtO O Br S N N HO N HO N O N OH HO O H HN N Tos N O Br S
O O O Br S N OH EtO O O NH N O N OH HO N HO S
Why is Molecular Framework Diversity Important?
Different molecular frameworks impart different activities
OH BzO H
HO O O Me HO O O Me OH O O OH HOH Me paeoniflorin OMe ON Anti-inflamatory activity H O O H Ph H Ph Me OMe O OH N O Me B O OH Me Me OMe Me Me
Me-CBS catalys rapamycin Catalytic enantioselective ketone reduction Immunosuppressant Molecular Framework Diversification Strategies
• MF transformations: convert a single MF to another one
– Trans-annular reactions - intra-framework bond making • Trans-annular reactions in TOS – Fragmentation reactions - intra-framework bond breaking • Fragmentation reactions in TOS – Fragmentation-trans-annular reaction sequences in TOS – MF transformations in DOS • Diverse MF synthesis: synthesis of a large number of MFs
– Linear vs. scaffold based library synthesis – Substrate controlled MF synthesis-folding pathways – Reagent controlled MF synthesis-branching pathways
Transannular Reactions in TOS
• Transannular Michael reaction
H H 0.1 eq PhSNa O O THF, reflux, 12h 93% H H O O- SPh H H PhS H
- H O O O O SPh - 1 bond H PhS H 3 stereocenters vicinal chiral quaternary centers H. W. Moore Org. Lett. 1999, 1, 375 Fragmentation Reaction
• Molecular framework diversification
OR Nu OR Nu
O Et2AlCl, DCM H H + R 4h, 25ºC H H 5 O O O R5
retro Diels-Alder OR Nu fragmentation RO H 130ºC, neat H Nu O Ar atm. O O
R5 retro-DA
E. Winterfeldt et al., Chimia 1993, 47, 39
Fragmentation Reactions in DOS
• Molecular framework diversity
O OR O Nu R H H Nu 5
H H O O 175 compounds OR Nu OR
H Nu Δ H O O O O R5 R5 5250 compounds 5250 compounds Examples of Diversity from Biosynthesis
Terpene Diversity
• Wide structural diversity and topological complexity in terpenoid molecules originate with isopentenyl pyrophosphate (IPP) and dimethallyl pyrophosphate (DMAPP) AcO O Ph O MeOH Ph O OH HO O HO PhCO2 O OAc HO O Taxol O O t-Bu O HO HO O Cholesterol O Ginkolide B OPP+ OPP IPP DMAPP
O OH O HN HO O CO H 2 Me S-CoA proto-Daphniphylline Gibberellic acid acetyl-coenzyme A Biosynthesis of IPP and DMAPP I
• All terpenes derived from IPP and DMAPP – Isopentenyl pyrophosphate (IPP) and dimethallyl pyrophosphate (DMAPP) biosynthesized from acetyl coenzyme A
O O O OO OOMe OH Me S-CoA Me S-CoA Me S-CoA Me S-CoA O S-CoA acetyl-CoA HMG-CoA acetyl coenzyme A thiolase synthetase HMG-CoA Claisen Aldol
O Me OH O Me OPP Me NADPH ATP [-CO2] OPP HMG-CoA O OH O OPP [-OPP] reductase mevalonic acid IPP
Biosynthesis of IPP and DMAPP II
• Stereospecific removal of the pro-R proton in IPP results in all-trans poly- isoprenylated chains
+ H Me Me isomerase ionize PPO Me OPP Me OPP H Me R HS DMAPP B IPP
Me IPP
OPP PPO Me H Me Me R HS B Me OPP TERPENES Me prenyl transferase(s) geranyl pyrophosphate Diversity Via Multiple Cyclization Pathways
• Diverse and complex terpene structures are obtained via a wide range of cyclization/rearrangement pathways – Terpene cyclase proteins mediate the cyclization process
O O O
OH
Me
OPP
Me Me Geranyl pyrophosphate
O
O O
Taxol Biosynthesis
• Taxol skeleton produced by taxadiene synthase mediated cyclization of geranylgeranyl pyrophosphate (diterpene) – Initial folding /cyclization events leading to the formation of the cationic macrocycle are enzyme mediated (reagent control). Subsequent intramolecular proton transfer is under substrate control
Me H Me Me Taxadiene Me synthase Me OPP AcO Me O oxidative MeOH modification Me RO Me
HO O OBz OAc Three stereocenters and three rings from GGPP! Taxol Williams, R.M., et al. Chem. Biol. 2000, 7, 969-976 Modular Polyketide Synthases
• To make chains of non-repeating units, modular PKSs have a separate set of FAS-like domains (modules) dedicated to each chain extension
LOAD MODULE 1 MODULE 1 MODULE 1
KR KR KR AT ACP KS AT ACP KS AT ACP KS AT ACP 2 3 CoA-S 1 S SH S S SH S O O O O O -CO Me Me O O Me Me 2 Me Propionyl CoA CO2H O CoA-S OH Me Me Methylmalonyl CoA 1) The AT of the load module loads the KS of module 1 with propionyl CoA MODULE 1 MODULE 2 2) The ACP is loaded with methyl malonyl CoA KR by the AT of module 1 KS AT ACP KS etc. 3) Decarboxylation and attack on the KS-bound 4 5 propionate gives the extended β-ketothioester SH S S O O 4) The KR reduces the β-ketothioester NADPH Me Me OH OH 5) No more reductive modules are present, so Me Me the chain is transferred to module 2
Erythromycin Biosynthesis
• Three large modular PKS enzymes (DEBS 1-3), encoded by eryAI, eryAII, and eryAIII, assemble the polyketide chain that forms the core of erythromycin
• each module performs one chain extension
O Me Me
Me Me TE OH cyclizes Me O OH
O OH Me 6-Deoxyerythronolide B
Staunton, J. Chem. Rev. 1997, 97, 2611. Erythromycin: DEBS Engineering - Deletions
O •Normal: Me Me
Me Me OH Me O OH
O OH • 80 aa deletion in the module 5 ketoreductase of DEBS Me 3: 6-Deoxyerythronolide B O Me Me
Me Me OH Me O O
O OH • Mutation in the NADPH-binding site of the module 4 Me enoylreductase of DEBS 2: O Me Me
Me Me OH Me O OH
O OH Me Pieper, R.; Kao, C.; Khosla, C.; Luo, G. Cane, D. E. Chem. Soc. Rev. 1996, 297. and refs therein
Erythromycin: DEBS Engineering - TE Transposition
O •Normal: Me Me Me Me OH Me O OH
O OH Me 6-Deoxyerythronolide B • Fusion of TE (from DEBS 3) onto DEBS 1:
OH Me Me
O O • Fusion of TE (from DEBS 3) onto DEBS 2:
O Me Me
Me Me OH O O OH Me Pieper, R.; Kao, C.; Khosla, C.; Luo, G. Cane, D. E. Chem. Soc. Rev. 1996, 297. and refs therein Erythromycin Biosynthesis
• A sequence of tailoring enzymes further functionalizes 6-DEB to give erythromycin A
O O Me Me NMe2 OH OH Me Me HO O Me + Me Me Me OH OH OH Selective Me O hydroxylation OH Me OH Me Me O OH Me D-desosamine L-mycarose O OH O OH Glycosyl- O OH transferases Me Me 6-Deoxyerythronolide B O O Me Me Me Me HO OH OH Selective Me Me OH Me NMe OH Me hydroxylation and 2 NMe2 Me HO Me HO methylation O O O Me O O O Me O O OMe O O OH Me Me Me Me OH OH O O Me Me Erythromycin A
Staunton, J. Chem. Rev. 1997, 97, 2611.
Construction of Polypropionates - Nature, PKS
• 4 reagents reductions • Complete reagent claisen condensations Me Me (enzyme)-based control O OH OH OH O O Me
HO recombinant Me Me Me Me Et Me PKS enzymes Premonensin O Me Me S-CoA OH OH OH OH + MeO C Me 2 O O H H MeMe Me Me Me Me Me S-CoA Zincophorin Me
OH OH OAc OMe
HO2C CHO Me Me Me Me Me
Rifamycin S (aliphatic fragment) Construction of Polypropionates - Laboratory
• Many reactions directed • Heavy reliance reduction aldol aldol Me Me on substrate- O OH OH OH O O Me based control HO makes each Me Me Me Me Et Me molecule a new alkylation alkylation Premonensin problem Ref. A hydroboration hetero-D.A. grignard Me addition OH OH OH OH Sigma-Aldrich Ref. B MeO C Me Catalog 2 O H H MeMe Me Me Me Me Me Ref. C hetero-D.A. Zincophorin epoxide Wittig; opening epoxidation hydroboration OH OH OAc OMe
HO2C CHO
Me Me Me Me Me allylation A: Evans, D. A.; DiMare, M. J. Am. Chem. Soc. 1986, 108, 2476. B: Danishevsky, S. J.; Selnick, H. G.; DeNinno, M. P.; Zelle, R. E. J. Am. Chem. Rifamycin S Wittig; Soc. 1987, 109, 1572. hydroboration C: Nagoaka, H.; Rutsch, W.; Schmid, G.; Iio, H.; Johnson, M. R.; Kishi, Y. J. Am. (aliphatic fragment) Chem. Soc. 1980, 102, 7962.